CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority from Provisional US Patent Application 63/326,883 to Merfeld, filed April 3, 2022, and entitled "Therapeutic polypseudorotaxane hydrogels," which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] Some implementations of the present invention relate in general to treatment of pathological central nervous system conditions, such as traumatic injury or neurodegenerative disease. More specifically, some implementations of the present invention relate to uses of hydrogels in the treatment of such conditions.

BACKGROUND

[0003] Traumatic injury to the central nervous system (CNS), such as traumatic brain injury (TBI) initially involves a mechanical force that causes tissue (e.g., neural tissue) damage. This can disrupt homeostasis, resulting in pathological cellular responses that can cause further damage, and/or prevent neural repair. Reactive astrogliosis, especially in more severe injuries, may inhibit neuronal growth at the injury site. For example, reactive astrocytes may form an astrocytic scar that forms a barrier around the injury site. While this barrier may beneficially serve to moderate and/or isolate the inflammatory response, it may also detrimentally inhibit influx of neural stem cells (NSC) and/or neuronal growth. (Burda et al. (2016) Experimental neurology, 275, 305-315.)

[0004] Rotaxanes and pseudorotaxanes are molecular complexes comprising a ring-shaped molecule that is threaded on an elongate molecule - in this context, often called an axle (Harrison, I. T., & Harrison, S. (1967). Journal of the American Chemical Society, 89(22), 5723-5724.). The ring-shaped molecule is inhibited from unthreading from the axle by steric effects or other molecular forces. For example, in rotaxanes, relatively bulky stoppers on the ends of the axle sterically block the ring-shaped molecule from unthreading from the axle.

[0005] Harada A. et al. ((1992) Nature 356:325) showed that rotaxanes may be formed using polymer axles comprising poly(ethylene oxide) (PEO) (also known as polyethylene glycol; PEG), with cyclodextrin (CD) serving as the ring-shaped molecule. Hydrogels based on PEO-CD compositions have been proposed for providing an injectable controlled-release matrix containing a drug (e.g., US 2002/0019369 to Li et al.). Li et al. describe that the properties of the hydrogels are tunable to affect the rate of drug release. For example, a PEO- CD hydrogel may be tailored to have a particular rate of bioabsorption. Li et al. further suggest, as example polymers for such a hydrogel, PEO block copolymers such as those with poly (propylene oxide) (PPO) including poloxamers.

SUMMARY OF THE INVENTION

[0006] This summary is meant to provide some examples and is not intended to be limiting of the scope of the invention in any way. For example, any feature included in an example of this summary is not required by the claims, unless the claims explicitly recite the features. Also, the features, components, steps, concepts, etc. described in examples in this summary and elsewhere in this disclosure can be combined in a variety of ways. Various features and steps as described elsewhere in this disclosure may be included in the examples summarized here.

[0007] There is therefore provided, in accordance with some implementations, a method, including: converting a polyp seudorotaxane hydrogel (PPRh) into a purified polypseudorotaxane hydrogel (pPPRh) by: dispersing the PPRh in an aqueous liquid, the PPRh including: axles of a polymer that includes ethylene oxide, alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and/or a-CD not threaded onto the axles (unthreaded a-CD); subsequently, accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD, such that the unthreaded a-CD is depleted in the pPPRh compared to in the PPRh; and/or using the pPPRh for manufacturing a medicament for Central Nervous System (CNS) introduction.

[0008] For some implementations, the pPPRh behaves as a Bingham plastic, and forming the pPPRh includes forming the pPPRh that behaves as a Bingham plastic.

[0009] For some implementations, the medicament is for piping into the brain, and using the pPPRh for manufacturing the medicament includes using the pPPRh for manufacturing the medicament that is for piping into the brain. [0010] For some implementations, the medicament is for treatment of traumatic brain injury, and using the pPPRh for manufacturing the medicament includes using the pPPRh for manufacturing the medicament that is for treatment of traumatic brain injury.

[0011] For some implementations, a concentration of unthreaded a-CD in the PPRh is cytotoxic, and converting the PPRh into the pPPRh includes depleting the unthreaded a-CD to a lower concentration that is less cytotoxic.

[0012] For some implementations, accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD includes centrifuging the aqueous liquid in which the PPRh is dispersed such that the axles and the threaded a-CD preferentially accrete as a pellet while the unthreaded a-CD preferentially remains in a supernatant.

[0013] For some implementations, dispersing the PPRh in the aqueous liquid includes dispersing the PPRh in the aqueous liquid while the aqueous liquid is at a temperature of between 50 and 70 degrees C.

[0014] For some implementations, in the pPPRh, the axles are substantially uncrosslinked.

[0015] For some implementations, the pPPRh has an storage modulus of 0.3- 1.5 kPa, and forming the pPPRh includes forming the pPPRh that has the storage modulus of 0.3- 1.5 kPa.

[0016] For some implementations, the method further includes forming the PPRh prior to dispersing the PPRh.

[0017] For some implementations, the PPRh contains unthreaded a-CD at greater than 200 mM, the pPPRh contains unthreaded a-CD at less than 20 mM, and converting the PPRh into the pPPRh includes converting the PPRh that contains unthreaded a-CD at greater than 200 mM into the pPPRh that contains unthreaded a-CD at less than 20 mM.

[0018] For some implementations, converting the PPRh into the pPPRh includes depleting the unthreaded a-CD in the PPRh by at least 90%.

[0019] For some implementations, the method further includes, subsequently to forming the pPPRh: holding the pPPRh at a temperature of between 50 and 80 degrees C, and/or while the pPPRh is at the temperature, stirring the pPPRh.

[0020] For some implementations: forming the pPPRh includes forming the pPPRh by accreting the axles and the threaded a-CD such that the pPPRh has a first storage modulus, the method further includes ceasing to hold and stir the pPPRh at the temperature, and/or holding and stirring the pPPRh includes holding and stirring the pPPRh for a duration such that, upon ceasing to hold and stir the pPPRh, the pPPRh has a second storage modulus that is greater than the first storage modulus.

[0021] For some implementations, the first storage modulus is 0.3-1 kPa.

[0022] For some implementations, the second storage modulus is approximately 1 kPa.

[0023] For some implementations, the temperature is between 50 and 70 degrees C, and holding the pPPRh at the temperature includes holding the pPPRh at the temperature that is between 50 and 70 degrees C.

[0024] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 2 and 10 min.

[0025] For some implementations, the method further includes dispersing cells within the pPPRh.

[0026] For some implementations, the cells are mesenchymal stem cells (MSC), and dispersing the cells within the pPPRh includes dispersing the MSC within the pPPRh.

[0027] For some implementations, dispersing the cells within the pPPRh includes: introducing the cells into the pPPRh; and/or subsequently, mixing the pPPRh containing the cells.

[0028] For some implementations, introducing the cells to the pPPRh includes depositing, distributed throughout the pPPRh, multiple deposits of a suspension of the cells.

[0029] For some implementations, depositing the multiple deposits includes depositing the multiple deposits using a bioprinter.

[0030] For some implementations, mixing the pPPRh includes passing the pPPRh through a mixing nozzle.

[0031] For some implementations, mixing the pPPRh includes stirring the pPPRh.

[0032] There is further provided, in accordance with some implementations, a composition for introduction into a site in a central nervous system of a subject, the composition including a polypseudorotaxane hydrogel (PPRh) that includes alpha-cyclodextrin (a-CD) threaded onto uncrosslinked axles of a polymer that includes ethylene oxide.

[0033] For some implementations, the axles are axles of poly(ethylene oxide) (PEO).

[0034] For some implementations, the composition is for introduction into a central nervous system (CNS) of a subject.

[0035] For some implementations, the composition is for treatment of a traumatic central nervous system (CNS) injury.

[0036] For some implementations, the composition is for treatment of a central nervous system (CNS) disease.

[0037] For some implementations, the composition behaves as a Bingham plastic.

[0038] For some implementations, the composition behaves as a fluid gel.

[0039] For some implementations, the PPRh does not include more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0040] For some implementations, the PPRh does not include more than 10 mM unthreaded a-CD.

[0041] For some implementations, the PPRh does not include more than 5 mM unthreaded a-CD.

[0042] For some implementations, the PPRh does not include more than 1 mM unthreaded a-CD.

[0043] For some implementations, the PPRh is substantially free of unthreaded a-CD.

[0044] For some implementations, the axles have unreacted methacrylate termini.

[0045] For some implementations, the composition does not include a photoinitiator.

[0046] For some implementations, the composition contains mesenchymal stem cells (MSC) suspended therein.

[0047] For some implementations, the composition contains MSC suspended therein at 50,000-200,000 cells per ml of PPRh.

[0048] For some implementations, the composition has a storage modulus of 0.5- 1.5 kPa.

[0049] For some implementations, the composition has a storage modulus of 0.6- 1.4 kPa.

[0050] For some implementations, the composition has a storage modulus of 0.7- 1.3 kPa. [0051] For some implementations, the composition has a storage modulus of 0.8- 1.2 kPa.

[0052] For some implementations, wherein the composition has a storage modulus of approximately 1 kPa.

[0053] There is further provided, in accordance with some implementations, a method, including: identifying a subject as having a pathological central nervous system (CNS) condition; and/or in response to the identifying, introducing, into a site in the CNS, a polypseudorotaxane hydrogel (PPRh) that includes alpha-cyclodextrin (a-CD) threaded onto axles of a polymer that includes ethylene oxide, the axles being substantially uncrosslinked.

[0054] For some implementations, the PPRh behaves as a Bingham plastic, and introducing the PPRh includes introducing the PPRh that behaves as a Bingham plastic.

[0055] For some implementations, the PPRh behaves as a fluid gel, and introducing the PPRh includes introducing the PPRh that behaves as a fluid gel.

[0056] For some implementations, introducing the PPRh includes piping the PPRh into the site.

[0057] For some implementations, introducing the PPRh includes squeezing the PPRh through a nozzle into the site.

[0058] For some implementations, introducing the PPRh includes introducing the PPRh via an access route into the subject, and the method further includes closing the access route while the axles remain substantially uncrosslinked.

[0059] For some implementations, the axles have methacrylate termini, introducing the PPRh includes introducing the PPRh via an access route into the site, and the method further includes closing the access route while the methacrylate termini remain substantially unreacted.

[0060] For some implementations, the subject is a patient, introducing the PPRh includes introducing the PPRh at a medical facility, and the method further includes discharging the patient from the medical facility while the axles remain substantially uncrosslinked.

[0061] For some implementations, the axles have methacrylate termini, the subject is a patient, introducing the PPRh includes introducing the PPRh at a medical facility, and the method further includes discharging the patient from the medical facility while the methacrylate termini remain substantially unreacted.

[0062] For some implementations, the pathological CNS condition is a traumatic CNS injury, and identifying the subject includes identifying the subject as having the traumatic CNS injury.

[0063] For some implementations, the pathological CNS condition is a CNS disease, and identifying the subject includes identifying the subject as having the CNS disease.

[0064] For some implementations, the PPRh has an storage modulus of 0.5- 1.5 kPa, and introducing the PPRh includes introducing the PPRh that has the storage modulus of 0.5- 1.5 kPa.

[0065] For some implementations, the PPRh has an storage modulus of 0.6- 1.4 kPa, and introducing the PPRh includes introducing the PPRh that has the storage modulus of 0.6- 1.4 kPa.

[0066] For some implementations, the PPRh has an storage modulus of 0.7- 1.3 kPa, and introducing the PPRh includes introducing the PPRh that has the storage modulus of 0.7- 1.3 kPa.

[0067] For some implementations, the PPRh has an storage modulus of 0.8- 1.2 kPa, and introducing the PPRh includes introducing the PPRh that has the storage modulus of 0.8- 1.2 kPa.

[0068] For some implementations, the PPRh has an storage modulus of approximately 1 kPa, and introducing the PPRh includes introducing the PPRh that has the storage modulus of approximately 1 kPa.

[0069] For some implementations: the PPRh includes less than 20 mM a-CD that is not threaded on the axles (unthreaded a-CD), and/or introducing the PPRh includes introducing the PPRh that includes less than 20 mM unthreaded a-CD.

[0070] For some implementations: the PPRh includes less than 10 mM unthreaded a-CD, and/or introducing the PPRh includes introducing the PPRh that includes less than 10 mM unthreaded a-CD. [0071] For some implementations, the PPRh contains cells dispersed therewithin, and introducing the PPRh includes introducing the PPRh that contains the cells dispersed therewithin.

[0072] For some implementations, the cells are mesenchymal stem cells (MSC), and introducing the PPRh includes introducing the PPRh that contains the MSC dispersed therewithin.

[0073] For some implementations, the method further includes introducing the cells into the PPRh, and subsequently mixing the PPRh containing the cells.

[0074] For some implementations, introducing the cells into the PPRh includes depositing, distributed throughout the PPRh, multiple deposits of a suspension of the cells.

[0075] For some implementations, depositing the multiple deposits includes depositing the multiple deposits using a bioprinter.

[0076] For some implementations, mixing the pPPRh includes passing the pPPRh through a mixing nozzle.

[0077] For some implementations, mixing the pPPRh includes stirring the pPPRh.

[0078] For some implementations, the method further includes, subsequently to introducing the PPRh, crosslinking the axles at a surface of the PPRh to form a cap beneath which the axles remaining uncrosslinked.

[0079] For some implementations, crosslinking the axles at the surface includes crosslinking the axles at the surface by applying ultraviolet light to the surface.

[0080] For some implementations, introducing the PPRh includes introducing the PPRh via an access route into the subject, and the method further includes closing the access route while the axles below the cap remain uncrosslinked.

[0081] For some implementations, the method further includes creating the access route.

[0082] For some implementations, the subject is a patient, introducing the PPRh includes introducing the PPRh at a medical facility, and the method further includes discharging the subject from the medical facility while the axles below the cap remain uncrosslinked.

[0083] There is further provided, in accordance with some implementations, a method, including: dispersing, in an aqueous liquid, a polypseudorotaxane hydrogel (PPRh) that includes: axles of a polymer that includes ethylene oxide, a cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and/or a-CD not threaded onto the axles (unthreaded a-CD), wherein dispersing the PPRh in the aqueous liquid includes dissolving the unthreaded a-CD in the aqueous liquid; and/or subsequently, by accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD, forming a purified PPRh (pPPRh) in which unthreaded a-CD is depleted compared to in the PPRh.

[0084] For some implementations, forming the pPPRh includes forming the pPPRh without crosslinking the axles.

[0085] For some implementations, the pPPRh is for use as a medicament.

[0086] For some implementations, the pPPRh behaves as a Bingham plastic, and forming the pPPRh includes forming the pPPRh that behaves as a Bingham plastic.

[0087] For some implementations, the pPPRh behaves as a fluid gel, and forming the pPPRh includes forming the pPPRh that behaves as a fluid gel.

[0088] For some implementations, dispersing the PPRh in the aqueous liquid includes dispersing the PPRh in the aqueous liquid while the aqueous liquid is at a temperature of between 50 and 70 degrees C.

[0089] For some implementations, in the PPRh, the axles are substantially uncrosslinked.

[0090] For some implementations, in the pPPRh, the axles are substantially uncrosslinked.

[0091] For some implementations, the pPPRh has an storage modulus of 0.3- 1.5 kPa, and forming the pPPRh includes forming the pPPRh that has the storage modulus of 0.3- 1.5 kPa.

[0092] For some implementations, forming the pPPRh includes forming the pPPRh without applying ultraviolet light to the pPPRh.

[0093] For some implementations, each of the axles has methacrylate termini, and forming the pPPRh includes forming the pPPRh in which each of the axles has methacrylate termini.

[0094] For some implementations, forming the pPPRh includes forming the pPPRh without including a photoinitiator in the pPPRh.

[0095] For some implementations: the pPPRh includes no photoinitiator, and/or the method further includes introducing, into a subject, the pPPRh that includes no photoinitiator.

[0096] For some implementations, the axles are axles of polyethylene oxide (PEO), and forming the pPPRh includes forming the pPPRh in which the axles are axles of PEO.

[0097] For some implementations, the axles are axles of a block copolymer, and forming the pPPRh includes forming the pPPRh in which the axles are axles of the block copolymer.

[0098] For some implementations, the method further includes forming the PPRh prior to dispersing the PPRh.

[0099] For some implementations, the method further includes, subsequently to forming the pPPRh: holding the pPPRh at a temperature of between 50 and 80 degrees C, and/or while the pPPRh is at the temperature, stirring the pPPRh.

[0100] For some implementations: forming the pPPRh includes forming the pPPRh by accreting the axles and the threaded a-CD such that the pPPRh has a first storage modulus, the method further includes ceasing to hold and stir the pPPRh at the temperature, and/or holding and stirring the pPPRh includes holding and stirring the pPPRh for a duration such that, upon ceasing to hold and stir the pPPRh, the pPPRh has a second storage modulus that is greater than the first storage modulus.

[0101] For some implementations, the first storage modulus is 0.3-1 kPa.

[0102] For some implementations, the second storage modulus is 0.5- 1.5 kPa.

[0103] For some implementations, the second storage modulus is 0.6- 1.4 kPa.

[0104] For some implementations, the second storage modulus is 0.7- 1.3 kPa.

[0105] For some implementations, the second storage modulus is 0.8- 1.2 kPa.

[0106] For some implementations, the second storage modulus is approximately 1 kPa.

[0107] For some implementations, the temperature is between 50 and 70 degrees C, and holding the pPPRh at the temperature includes holding the pPPRh at the temperature that is between 50 and 70 degrees C. [0108] For some implementations, the temperature is between 55 and 65 degrees C, and holding the pPPRh at the temperature includes holding the pPPRh at the temperature that is between 55 and 65 degrees C.

[0109] For some implementations, the temperature is between 58 and 62 degrees C, and holding the pPPRh at the temperature includes holding the pPPRh at the temperature that is between 58 and 62 degrees C.

[0110] For some implementations, the temperature is approximately 60 degrees C, and holding the pPPRh at the temperature includes holding the pPPRh at the temperature that is approximately 60 degrees C.

[0111] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 2 and 60 min.

[0112] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 2 and 30 min.

[0113] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 5 and 25 min.

[0114] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 10 and 25 min.

[0115] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 10 and 15 min.

[0116] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 15 and 25 min.

[0117] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 2 and 10 min.

[0118] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 2 and 5 min.

[0119] For some implementations, holding the solution at the temperature includes holding the solution at the temperature for a duration of between 5 and 10 min.

[0120] For some implementations, the method further includes introducing the pPPRh into a central nervous system (CNS) of a subject.

[0121] For some implementations, introducing the pPPRh into the CNS includes piping the pPPRh into the CNS. [0122] For some implementations, introducing the pPPRh into the CNS includes introducing the pPPRh into a brain of the subject.

[0123] For some implementations, introducing the pPPRh into the CNS includes introducing the pPPRh into the CNS in response to identifying the subject as having a traumatic CNS injury.

[0124] For some implementations, accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD includes centrifuging the aqueous liquid in which the PPRh is dispersed such that the axles and the threaded a-CD preferentially form a pellet while the unthreaded a-CD preferentially remains in a supernatant.

[0125] For some implementations, centrifuging the aqueous liquid includes centrifuging the aqueous liquid at 100-200 x g.

[0126] For some implementations, centrifuging the aqueous liquid includes centrifuging the aqueous liquid for 2-10 min.

[0127] For some implementations, the method further includes dispersing cells within the pPPRh.

[0128] For some implementations, the cells are mesenchymal stem cells (MSC), and dispersing the cells within the pPPRh includes dispersing the MSC within the pPPRh.

[0129] For some implementations, dispersing the cells within the pPPRh includes: introducing the cells into the pPPRh; and/or subsequently, mixing the pPPRh containing the cells.

[0130] For some implementations, introducing the cells to the pPPRh includes depositing, distributed throughout the pPPRh, multiple deposits of a suspension of the cells.

[0131] For some implementations, depositing the multiple deposits includes depositing the multiple deposits using a bioprinter.

[0132] For some implementations, mixing the pPPRh includes passing the pPPRh through a mixing nozzle.

[0133] For some implementations, mixing the pPPRh includes stirring the pPPRh.

[0134] For some implementations, the PPRh contains unthreaded a-CD at greater than 50 mM, and dispersing the PPRh in the aqueous liquid includes dispersing, in the aqueous liquid, the PPRh that contains unthreaded a-CD at greater than 50 mM. [0135] For some implementations, the PPRh contains unthreaded a-CD at greater than 100 mM, and dispersing the PPRh in the aqueous liquid includes dispersing, in the aqueous liquid, the PPRh that contains unthreaded a-CD at greater than 100 mM.

[0136] For some implementations, the PPRh contains unthreaded a-CD at greater than 200 mM, and dispersing the PPRh in the aqueous liquid includes dispersing, in the aqueous liquid, the PPRh that contains unthreaded a-CD at greater than 200 mM.

[0137] For some implementations, the pPPRh contains unthreaded a-CD at less than 20 mM, and forming the pPPRh includes forming the pPPRh that contains unthreaded a-CD at less than 20 mM.

[0138] For some implementations, the pPPRh contains unthreaded a-CD at less than 10 mM, and forming the pPPRh includes forming the pPPRh that contains unthreaded a-CD at less than 10 mM.

[0139] For some implementations, the pPPRh contains unthreaded a-CD at less than 5 mM, and forming the pPPRh includes forming the pPPRh that contains unthreaded a-CD at less than 5 mM.

[0140] For some implementations, the pPPRh contains substantially no unthreaded a-CD, and forming the pPPRh includes forming the pPPRh that contains substantially no unthreaded a-CD.

[0141] There is further provided, in accordance with some implementations, a method, including: holding, at a temperature of between 50 and 70 degrees C, a polyp seudorotaxane hydrogel (PPRh) that includes alpha-cyclodextrin (a-CD) threaded onto axles of a polymer that includes ethylene oxide, the axles being uncrosslinked; and/or while the PPRh remains at the temperature, stirring the PPRh.

[0142] For some implementations, holding the PPRh at the temperature includes holding the PPRh at the temperature until the PPRh behaves as a Bingham plastic.

[0143] For some implementations, holding the PPRh at the temperature includes holding the PPRh at the temperature until the PPRh behaves as a fluid gel.

[0144] For some implementations, the method further includes, prior to holding the PPRh at the temperature, forming the PPRh. [0145] There is further provided, in accordance with some implementations, uncrosslinked polypseudorotaxane hydrogel (uPPRh) for use as a medicament.

[0146] For some implementations, the uPPRh includes axles of poly(ethylene oxide) (PEO).

[0147] For some implementations, the uPPRh is for introduction into a central nervous system (CNS) of a subject.

[0148] For some implementations, the uPPRh is for treatment of a traumatic central nervous system (CNS) injury.

[0149] For some implementations, the uPPRh is for treatment of a central nervous system (CNS) disease.

[0150] For some implementations, the uPPRh behaves as a Bingham plastic.

[0151] For some implementations, the uPPRh behaves as a fluid gel.

[0152] For some implementations: the uPPRh includes: axles of a polymer that includes ethylene oxide, and/or alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and/or the uPPRh does not include more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0153] For some implementations, the uPPRh does not include more than 10 mM unthreaded a-CD.

[0154] For some implementations, the uPPRh does not include more than 5 mM unthreaded a-CD.

[0155] For some implementations, the uPPRh does not include more than 1 mM unthreaded a-CD.

[0156] For some implementations, the uPPRh is substantially free of unthreaded a-CD.

[0157] For some implementations, the uPPRh includes polymer axles that have unreacted methacrylate termini.

[0158] For some implementations, the uPPRh does not include a photoinitiator.

[0159] For some implementations, the uPPRh contains mesenchymal stem cells (MSC) suspended therein. [0160] For some implementations, the uPPRh contains MSC suspended therein at 50,000- 200,000 cells per ml of uPPRh.

[0161] For some implementations, the uPPRh has a storage modulus of 0.5- 1.5 kPa.

[0162] For some implementations, the uPPRh has a storage modulus of 0.6- 1.4 kPa.

[0163] For some implementations, the uPPRh has a storage modulus of 0.7- 1.3 kPa.

[0164] For some implementations, the uPPRh has a storage modulus of 0.8- 1.2 kPa.

[0165] For some implementations, the uPPRh has a storage modulus of approximately 1 kPa.

[0166] There is further provided, in accordance with some implementations, uncrosslinked polypseudorotaxane hydrogel (uPPRh) for use in the treatment of a pathological Central Nervous System (CNS) condition.

[0167] For some implementations, the uPPRh includes axles of poly(ethylene oxide) (PEO).

[0168] For some implementations, the uPPRh is for introduction into the CNS of a subject.

[0169] For some implementations, the pathological CNS condition is a traumatic CNS injury.

[0170] For some implementations, the pathological CNS condition is a CNS disease.

[0171] For some implementations, the uPPRh behaves as a Bingham plastic.

[0172] For some implementations, the uPPRh behaves as a fluid gel.

[0173] For some implementations: the uPPRh includes: axles of a polymer that includes ethylene oxide, and/or alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and/or the uPPRh does not include more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0174] For some implementations, the uPPRh does not include more than 10 mM unthreaded a-CD.

[0175] For some implementations, the uPPRh does not include more than 5 mM unthreaded a-CD.

[0176] For some implementations, the uPPRh does not include more than 1 mM unthreaded a-CD. [0177] For some implementations, the uPPRh is substantially free of unthreaded a-CD.

[0178] For some implementations, the uPPRh includes polymer axles that have unreacted methacrylate termini.

[0179] For some implementations, the uPPRh does not include a photoinitiator.

[0180] For some implementations, the uPPRh contains mesenchymal stem cells (MSC) suspended therein.

[0181] For some implementations, the uPPRh contains MSC suspended therein at 50,000- 200,000 cells per ml of uPPRh.

[0182] For some implementations, wherein the uPPRh has a storage modulus of 0.5- 1.5 kPa.

[0183] For some implementations, the uPPRh has a storage modulus of 0.6- 1.4 kPa.

[0184] For some implementations, the uPPRh has a storage modulus of 0.7- 1.3 kPa.

[0185] For some implementations, the uPPRh has a storage modulus of 0.8- 1.2 kPa.

[0186] For some implementations, the uPPRh has a storage modulus of approximately 1 kPa.

[0187] There is further provided, in accordance with some implementations, uncrosslinked polypseudorotaxane hydrogel (uPPRh) for use in the treatment of a traumatic Central Nervous System (CNS) injury.

[0188] For some implementations, uPPRh includes axles of poly(ethylene oxide) (PEO).

[0189] For some implementations, the uPPRh is for introduction into the CNS of a subject.

[0190] For some implementations, the uPPRh behaves as a Bingham plastic.

[0191] For some implementations, the uPPRh behaves as a fluid gel.

[0192] For some implementations: the uPPRh includes: axles of a polymer that includes ethylene oxide, and/or alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and/or the uPPRh does not include more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0193] For some implementations, the uPPRh does not include more than 10 mM unthreaded a-CD. [0194] For some implementations, the uPPRh does not include more than 5 mM unthreaded a-CD.

[0195] For some implementations, the uPPRh does not include more than 1 mM unthreaded a-CD.

[0196] For some implementations, the uPPRh is substantially free of unthreaded a-CD.

[0197] For some implementations, the uPPRh includes polymer axles that have unreacted methacrylate termini.

[0198] For some implementations, the uPPRh does not include a photoinitiator.

[0199] For some implementations, the uPPRh contains mesenchymal stem cells (MSC) suspended therein.

[0200] For some implementations, the uPPRh contains MSC suspended therein at 50,000- 200,000 cells per ml of uPPRh.

[0201] For some implementations, the uPPRh has a storage modulus of 0.5- 1.5 kPa.

[0202] For some implementations, the uPPRh has a storage modulus of 0.6- 1.4 kPa.

[0203] For some implementations, the uPPRh has a storage modulus of 0.7- 1.3 kPa.

[0204] For some implementations, the uPPRh has a storage modulus of 0.8- 1.2 kPa.

[0205] For some implementations, the uPPRh has a storage modulus of approximately 1 kPa.

[0206] There is further provided, in accordance with some implementations, apparatus, including: a polypseudorotaxane hydrogel (PPRh) that includes alpha-cyclodextrin (a-CD) threaded onto uncrosslinked axles of a polymer that includes ethylene oxide; and/or an applicator, configured to introduce the PPRh into a site in a central nervous system of a subject.

[0207] For some implementations, the PPRh is disposed within the applicator.

[0208] For some implementations, the PPRh behaves as a Bingham plastic.

[0209] For some implementations, the PPRh behaves as a fluid gel.

[0210] For some implementations, the applicator has a nozzle, and is configured to squeeze the PPRh through the nozzle and into the site. [0211] For some implementations, the system further includes a capping tool that includes an ultraviolet (UV) light source, the tool configured to apply a dose of UV light to a surface of the PPRh at the site, the dose of UV light having a wavelength, duration, and intensity adapted to crosslink the axles at the surface to form a cap beneath which the axles remain uncrosslinked.

[0212] For some implementations, the applicator includes an ultraviolet (UV) light source, the applicator being configured to apply a dose of UV light to a surface of the PPRh at the site, the dose of UV light having a wavelength, duration, and intensity adapted to crosslink the axles at the surface to form a cap beneath which the axles remain uncrosslinked.

[0213] For some implementations, the PPRh is for treatment of a traumatic central nervous system (CNS) injury.

[0214] For some implementations, the PPRh is for treatment of a central nervous system (CNS) disease.

[0215] For some implementations, the PPRh does not include more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0216] For some implementations, the PPRh does not include more than 10 mM unthreaded a-CD.

[0217] For some implementations, the PPRh does not include more than 5 mM unthreaded a-CD.

[0218] For some implementations, the PPRh does not include more than 1 mM unthreaded a-CD.

[0219] For some implementations, the PPRh is substantially free of unthreaded a-CD.

[0220] For some implementations, the axles have unreacted methacrylate termini.

[0221] For some implementations, the PPRh does not include a photoinitiator.

[0222] For some implementations, the PPRh contains mesenchymal stem cells (MSC) suspended therein.

[0223] For some implementations, the PPRh contains MSC suspended therein at 50,000- 200,000 cells per ml of PPRh.

[0224] For some implementations, the PPRh has a storage modulus of 0.5- 1.5 kPa.

[0225] For some implementations, the PPRh has a storage modulus of 0.6- 1.4 kPa. [0226] For some implementations, the PPRh has a storage modulus of 0.7- 1.3 kPa.

[0227] For some implementations, the PPRh has a storage modulus of 0.8- 1.2 kPa.

[0228] For some implementations, the PPRh has a storage modulus of approximately 1 kPa.

[0229] There is further provided, in accordance with some implementations, a method, including: depositing, into a hydrogel, multiple deposits of a suspension of cells such that the multiple deposits are distributed throughout the hydrogel; and/or subsequently, mixing the hydrogel such that each of the deposits disperses.

[0230] For some implementations, depositing the multiple deposits includes depositing the multiple deposits using a bioprinter.

[0231] For some implementations, mixing the hydrogel includes passing the hydrogel through a mixing nozzle.

[0232] For some implementations, mixing the hydrogel includes stirring the hydrogel.

[0233] For some implementations, the hydrogel behaves as a Bingham plastic, and mixing the hydrogel includes mixing the hydrogel that behaves as a Bingham plastic.

[0234] For some implementations, the hydrogel behaves as a fluid gel, and mixing the hydrogel includes mixing the hydrogel that behaves as a fluid gel.

[0235] For some implementations: the cells are mesenchymal stem cells (MSC), and/or depositing the multiple deposits includes depositing the multiple deposits of the suspension of MSC.

[0236] For some implementations, depositing the multiple deposits includes depositing multiple deposits that each contain 500-2000 of the cells.

[0237] For some implementations, depositing the multiple deposits includes depositing multiple deposits that each has a volume of 5-20 microliters.

[0238] For some implementations, depositing the multiple deposits includes depositing 50- 200 deposits per 1 ml of the hydrogel.

[0239] For some implementations, each of the deposits is a discrete punctate deposit, and depositing the multiple deposits into the hydrogel includes depositing the multiple discrete punctate deposits into the hydrogel. [0240] For some implementations, each of the deposits is an elongate deposit, and depositing the multiple deposits into the hydrogel includes depositing the multiple elongate deposits into the hydrogel.

[0241] For some implementations, the method further includes, subsequently to mixing the hydrogel, introducing the hydrogel into a central nervous system (CNS) of a subject.

[0242] For some implementations, introducing the hydrogel into the CNS includes piping the hydrogel into the CNS.

[0243] For some implementations, introducing the hydrogel into the CNS includes introducing the hydrogel into a brain of the subject.

[0244] For some implementations, introducing the hydrogel into the CNS includes introducing the hydrogel into the CNS in response to identifying the subject as having a traumatic CNS injury.

[0245] For some implementations: the hydrogel has an storage modulus of 0.5-2 kPa, and/or depositing the multiple deposits into the hydrogel includes depositing the multiple deposits into the hydrogel that has the storage modulus of 0.5-2 kPa.

[0246] For some implementations: the hydrogel has a storage modulus of 0.5- 1.5 kPa, and/or depositing the multiple deposits into the hydrogel includes depositing the multiple deposits into the hydrogel that has the storage modulus of 0.5- 1.5 kPa.

[0247] For some implementations: the hydrogel has an storage modulus of 0.6- 1.3 kPa, and/or depositing the multiple deposits into the hydrogel includes depositing the multiple deposits into the hydrogel that has the storage modulus of 0.6- 1.3 kPa.

[0248] For some implementations: the hydrogel has a storage modulus of 0.8- 1.2 kPa, and/or depositing the multiple deposits into the hydrogel includes depositing the multiple deposits into the hydrogel that has the storage modulus of 0.8- 1.2 kPa.

[0249] For some implementations: the hydrogel has a storage modulus of approximately 1 kPa, and/or depositing the multiple deposits into the hydrogel includes depositing the multiple deposits into the hydrogel that has the storage modulus of approximately 1 kPa. [0250] For some implementations: the hydrogel is a polyp seudorotaxane hydrogel (PPRh) that includes alphacyclodextrin (a-CD) threaded on axles of a polymer that includes ethylene oxide, and/or depositing the deposits into the hydrogel includes depositing the deposits into the PPRh.

[0251] For some implementations: the axles are substantially uncrosslinked, depositing the deposits into the PPRh includes depositing the deposits into the PPRh in which the axles are uncrosslinked, and/or mixing the hydrogel includes mixing the PPRh in which the axles are substantially uncrosslinked.

[0252] For some implementations: the PPRh includes less than 20 mM a-CD that is not threaded on the axles (unthreaded a-CD), and/or depositing the deposits into the PPRh includes depositing the deposits into the PPRh that includes less than 20 mM unthreaded a-CD.

[0253] For some implementations: the PPRh includes less than 10 mM unthreaded a-CD, and/or depositing the deposits into the PPRh includes depositing the deposits into the PPRh that includes less than 10 mM unthreaded a-CD.

[0254] For some implementations: the PPRh includes less than 5 mM unthreaded a-CD, and/or depositing the deposits into the PPRh includes depositing the deposits into the PPRh that includes less than 5 mM unthreaded a-CD.

[0255] The present invention will be more fully understood from the following detailed description of applications thereof, taken together with the drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

[0256] Fig. 1 is a schematic representation of a proposed model of the relationship between storage modulus and cellular effects thereof;

[0257] Fig. 2 shows data from the examination of viability of primary hippocampal neurons; [0258] Fig. 3 shows data from the examination of expression of glial fibrillary acidic protein (GFAP) by hippocampal astrocytes;

[0259] Fig. 4 is a schematic illustration showing at least some steps in a technique for forming an implantable polyp seudorotaxane hydrogel PPRh, in accordance with some implementations ;

[0260] Fig. 5 shows data from an experiment in which mesenchymal stem cells (MSC) were mixed into uncrosslinked PPRh in which alpha-cyclodextrin (a-CD) had not been removed;

[0261] Fig. 6 shows data from an experiment in which a-CD was introduced into growth medium in which MSC were cultured;

[0262] Fig. 7 is a schematic illustration showing at least some steps in a technique for manufacturing an uncrosslinked PPRh that has a low prevalence of unthreaded a-CD, in accordance with some implementations;

[0263] Fig. 8 shows data from an experiment in which pPPRh was held at an elevated temperature, while stirring, for various durations;

[0264] Fig. 9 is a schematic illustration of a technique for distributing cells within a hydrogel, in accordance with some implementations; and

[0265] Figs. 10A-C and 11 A-C are schematic illustrations of a technique for using a purified PPRh, in accordance with some implementations.

DETAILED DESCRIPTION OF EMBODIMENTS

[0266] Reference is made to Figs. 1-3. Potentially beneficial effects of the introduction of polypseudorotaxane hydrogel (PPRh) into the central nervous system (CNS) have previously been described in US Patent Application Publication 2021/0023266 to Merfeld et al., which is incorporated herein by reference. Such benefits include promotion of neuronal maturation and inhibition of astrogliosis. Experiments described therein, as well as subsequent in vitro and in vivo experiments, indicate that both of these effects can be dependent on the elasticity (e.g. the storage modulus) of the PPRh.

[0267] Fig. 1 is a schematic representation of a proposed model of the relationship between storage modulus and these cellular effects. The beneficial enhancement, by PPRh, of neuronal maturation was found to decrease with increased storage modulus of the PPRh - e.g. as demonstrated by the data shown in Fig. 2. In contrast, the beneficial inhibition, by PPRh, of astrogliosis was found to increase with increased storage modulus of the PPRh - e.g. as demonstrated by the data shown in Fig. 3. Moreover, whereas the correlation between elasticity and decreased neuronal maturation was found to be somewhat linear, the correlation between elasticity and increased inhibition of astrogliosis was found to be somewhat bell-shaped.

[0268] The data shown in Figs. 2-3 were generated in vitro. 20,000 cells primary hippocampal neurons (Fig. 2) or hippocampal astrocytes (Fig. 3) from postnatal rat pup brains were seeded, separately, in 96 well plates. The cells were overlaid with a 1 mm thick disk of PPRh with a low storage modulus (roughly 1 kPa), medium storage modulus (roughly 3 kPa) or high storage modulus (roughly 7 kPa). A control group was overlaid with a 1 mm thick disk of agarose gel (3 percent). Additionally, another control group was provided in which no hydrogel was laid over the cells.

[0269] Rheological measurements were performed as follows: PPRhs were cut into disks with 20 mm diameter and a 1 mm thickness. Elastic (G') and loss (G") moduli were recorded with the frequency increased from 1 rad/s to 100 rad/s and a strain of 0.5 percent on a stress- controlled rheometer (TA instruments, DHR-2) with a 20-mm diameter parallel plate geometry and a measuring gap of 1 mm at room temperature. The averaged values at different frequencies were obtained by averaging the moduli measured from three PPRh- disks with each ratio. All the PPRhs possess frequency-independent elastic moduli.

[0270] Each of the above PPRhs comprises alpha-cyclodextrin (a-CD) molecules threaded onto crosslinked axles of PEO homopolymer, each axle being approximately 450 monomers long. However, for some implementations, other axles, such as axles of a block copolymer, may be used. The different elastic moduli of the various PPRh result from different a- CD:axle ratios within the PPRh, which itself derives from the a-CD:axle ratio in the starting solution from which the PPRh is formed. For example, the medium-elasticity PPRh is made from a starting solution having an a-CD: axle molar ratio of 118:1, a higher a-CD: axle ratio results in the high-elasticity PPRh, and a lower a-CD:axle ratio results in the low elasticity PPRh. It is understood that this effect is because (i) the a-CD:axle ratio in the starting solution correlates with the average number of a-CD that become threaded on each axle in the PPRh - known as the "threading ratio", and (ii) a higher threading ratio generates more attraction between axles, thereby increasing the storage modulus of the PPRh. Other than the differences in their a-CD:axle ratios, the three PPRhs have the same composition.

[0271] Each PPRh disk was immersed in 70 percent ethanol for 30 min, washed 3 times with phosphate-buffered saline (PBS), and soaked for 72 h in growth medium enriched with L- Lysine (20 microgram/ml). Each PPRh disk was then laid onto the seeded cells, and the cells were then incubated for 72 h in the presence (or absence) of the corresponding hydrogel, and were subsequently examined.

[0272] Fig. 2 shows data from the examination of the viability of the primary hippocampal neurons. This was measured using an XTT-based cell proliferation kit (Biological Industries Israel Beit HaEmek Ltd.; cat 20-300-1000).

[0273] In all cases except one, the viability of the cultured cells was at least slightly reduced by the presence of any of the gels - agarose or PPRh. However, the low-elasticity PPRh promoted the viability of neurons. That is, in the presence of the low-elasticity PPRh, the viability of cultured neurons was higher than in the control. It is therefore hypothesized by the inventor that introduction of a low-elasticity PPRh into the CNS (e.g. into the brain) may be useful in promoting neuronal recovery in mammalian (e.g., human) subjects.

[0274] Fig. 3 shows data from the examination of expression of glial fibrillary acidic protein (GFAP) by hippocampal astrocytes. GFAP is an intermediate filament protein expressed by several CNS cells, but is expressed particularly strongly by astrocytes. Expression of GFAP is further upregulated in activated astrocytes, and so can serve as an indicator of reactive astrogliosis.

[0275] GFAP expression was measured by staining cell cultures with fluorescent anti-GFAP antibody. Stained cultures were tile scanned using Zeiss fluorescence microscope, images were stitched, and GFAP fluorescence was analyzed using Imaris 9.2 software (Bitplane AG, Switzerland). For each image, 3-6 surface segments with similar surface size were positioned within areas where cell population is well established. Surface segments were then quantitatively analyzed.

[0276] All three PPRhs reduced maximal GFAP expression, but this reduction was greater for the medium- and high-elasticity PPRhs. Upregulated GFAP expression is associated with activated astrocytes. It is therefore hypothesized that reducing maximal expression of GFAP expression may be important in preventing and/or treating astrocyte-mediated pathologies.

[0277] It is therefore hypothesized that having a high degree of control of the elasticity of a PPRh may advantageously allow a PPRh to be manufactured that both (i) facilitates beneficial neuronal recovery and maturation, and (ii) inhibits detrimental astrogliosis. For example, such a PPRh may have an storage modulus that is sufficiently low to facilitate neuronal recovery and maturation, but sufficiently high to inhibit astrogliosis. This may be advantageous in treating a pathological CNS condition such as traumatic CNS injury (e.g. traumatic brain injury, TBI) or a CNS disease such as stroke, glioma (e.g., astrocytoma), or a neurodegenerative disorder (e.g. amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's Disease, and Parkinson's Disease). An example of such an storage modulus is indicated in Fig. 1 by an asterisk.

[0278] Reference is now made to Fig. 4, which is a schematic illustration showing at least some steps in a technique 10 for forming an implantable PPRh, in accordance with some implementations. Technique 10 includes (i) threading a-CD on the axles by combining, in solution, the axles and excess a-CD (step 12); (ii) subsequently, securing the threaded a-CD on the axles by cross-linking the axles - e.g. by applying ultraviolet light (step 14); and (iii) removing unthreaded a-CD by washing the PPRh in a solvent such as dimethyl sulfoxide (DMSO) (step 16). Unthreaded a-CD are a-CD that are disposed between the axles rather than being threaded on the axles, but that are nonetheless associated with other unthreaded a-CD, with the threaded a-CD, and/or with the axles. Unthreaded a-CD is represented by hexagons 18. In Fig. 4, crosslinking of the PPRh is represented by bolding of the box that represents the PPRh. The association of unthreaded a-CD with the threaded a-CD and/or with the axles may undesirably (and possibly unpredictably) affect the mechanical properties of the PPRh, such as by increasing its elasticity. At least some of this effect may be due to crystallization of the unthreaded a-CD.

[0279] For a PPRh that is to be introduced into the CNS to promote neuronal recovery and maturation, it may be advantageous to include particular cells, such as mesenchymal stem cells (MSC) within the PPRh. For example, a storage modulus optimized for promoting neuronal recovery and maturation may be lower than that optimized for inhibiting astrogliosis (e.g. see Fig. 1), and inclusion of MSC may allow a PPRh whose storage modulus is optimized for neuronal recovery and maturation to also have astrogliosisinhibiting properties.

[0280] Furthermore, it may be advantageous for the PPRh to be administered via a nozzle (e.g. piped or squeezed through the nozzle and/or injected into the subject), e.g. so as to introduce the PPRh via a small access opening, and/or so as to fill a lesion (which may not be regularly shaped, and which may include crevices) effectively.

[0281] Prior crosslinking of the axles may be unfavorable to nozzle administration and/or to mixing of cells into the PPRh because the crosslinking increases the behavior of the PPRh as an elastic solid with a relatively defined shape (e.g. similar to a set gelatin or agarose gel), such that nozzle administration and/or mixing may irreversibly break the PPRh. [0282] At least in light of the above disadvantages of crosslinking the PPRh, it is hypothesized that it may be advantageous to introduce cells into uncrosslinked PPRh, and/or to introduce PPRh into the CNS while uncrosslinked. However, this raises further challenges. In the technique described with reference to Fig. 4, crosslinking is performed prior to removing unthreaded a-CD in order to secure the a-CD on the axles, so that washing in DMSO does not unthread a-CD from the axles and effectively dissolve the PPRh.

[0283] Introducing the cells into uncrosslinked PPRh, then crosslinking the axles, and then performing a DMSO wash is unlikely to be effective because DMSO is toxic to cells.

[0284] Introducing the cells into uncrosslinked PPRh and simply leaving the unthreaded a- CD within the PPRh (whether or not the axles are crosslinked after the introduction of the cells) may also be suboptimal - at least for some implementations. As described hereinabove, unthreaded a-CD may have an undesirable effect on the mechanical properties of the PPRh. However, it has additionally been determined that the presence of unthreaded a-CD may be detrimental to cells disposed within the PPRh (e.g. MSC suspended in the PPRh) or adjacent to the PPRh (e.g. CNS tissue adjacent the site in which the PPRh is implanted). Figs. 5 and 6 show examples of data supporting this determination. It is hypothesized that, for some implementations, this may be due to the a-CD increasing the osmolarity of the liquid component (e.g. liquid phase) of the PPRh.

[0285] Fig. 5 shows data from an experiment in which MSCs were mixed into uncrosslinked PPRh in which a-CD had not been removed (PPRh+MSCs). A positive control group of MSC only (MSCs), and a negative control group of PPRh only (PPRh) were also included. After 3 or 18 hours of incubation, cell viability was measured using an XTT-based cell proliferation kit (Biological Industries Israel Beit HaEmek Ltd.; cat 20-300-1000). At both time points the cell viability in the PPRh+MSCs was significantly lower than the positive control group of MSC only, and was not significantly different from the negative control group of PPRh only.

[0286] Fig. 6 shows data from an experiment in which a-CD was introduced into the medium in which MSC had been cultured. 18 hours after the introduction of the a-CD, the viability of the MSC was tested. Whereas concentrations of up to 4mM were tolerated by the MSC, a significant drop in cell viability was observed at and beyond the next highest concentration - 20 mM. A negative control group containing no MSC was also included ("no MSC").

[0287] Thus, for at least some implementations, for mechanical and/or cell viability reasons, it may be undesirable to introduce cells (e.g. MSC) into uncrosslinked PPRh while leaving the unthreaded a-CD within the PPRh (whether or not the axles are crosslinked after the introduction of the cells). However, for reasons described hereinabove, the crosslink-then- wash technique shown in Fig. 4 may also be unsuitable.

[0288] Reference is now made to Fig. 7. In light of the above, a technique 20 has been developed for manufacturing an uncrosslinked PPRh that has a low prevalence of unthreaded a-CD - e.g. a concentration of less than 20 mM (e.g. less than 10 mM, e.g. less than 5 mM, e.g. less than 1 mM, e.g. substantially zero) unthreaded a-CD (e.g. in the liquid component of the PPRh). The technique advantageously involves substantially depleting the unthreaded a-CD from the uncrosslinked PPRh while substantially retaining the threaded a-CD threaded on the axles. That is, the technique preferentially removes unthreaded a-CD compared with threaded a-CD.

[0289] Fig. 7 is a schematic illustration showing at least some steps in technique 20, in accordance with some implementations. The a-CD is threaded onto the axles by combining, in solution, the axles and excess a-CD (step 22), resulting in uncrosslinked PPRh containing unthreaded a-CD (represented by hexagons 18). Due to the excess a-CD in the initial solution, it is estimated that the PPRh (e.g. the liquid component thereof) typically contains unthreaded a-CD at greater than 50 mM (e.g. at about 75 mM) - and possibly even greater - e.g. greater than 100 mM (such as greater than 200 mM). Step 22 and/or the PPRh resulting therefrom, may be similar (e.g. identical) to step 12 of technique 10 and/or the PPRh resulting therefrom. However, whereas in technique 10 the axles of the PPRh are then crosslinked in preparation of the DMSO wash to remove unthreaded a-CD, in technique 20 the axles are left uncrosslinked. This is possible because the removal of unthreaded a-CD (steps 24 & 26) is achieved by a means (described hereinbelow) that is hypothesized to be gentler on the "pseudorotaxanes" (the axles with a-CD threaded thereon) - e.g. sufficiently gentle that the threaded a-CD remain threaded on the axles despite the unthreaded a-CD being removed.

[0290] Step 24 includes dispersing the PPRh in an aqueous liquid (Aq) such as water, saline, phosphate-buffered saline (PBS), or a cell-growth medium. This dispersal is hypothesized to dissolve the unthreaded a-CD in the liquid while leaving the pseudorotaxanes intact (i.e. leaving the threaded a-CD threaded on the axles) - despite loosening the association between the pseudorotaxanes. For some implementations, this dispersal is extensive (e.g. the PPRh may appear to have dissolved in the liquid). For some implementations, this dispersal may be limited (e.g. the PPRh may merely be softened or loosened). In Fig. 7, dispersal of the PPRh is represented by the box that represents the PPRh being cloud-shaped. The volume of the liquid in which the PPRh is dispersed is typically at least 10 times the volume of the PPRh (e.g. may be 10-100 times, e.g. 30-70 times, such as approximately 50 times the volume of the PPRh), so as to significantly dilute the unthreaded a-CD throughout the volume. For some implementations, the liquid in which the PPRh is dispersed is at a temperature of between 50 and 70 degrees C.

[0291] Subsequently, the pseudorotaxanes (i.e. the axles and the threaded a-CD threaded thereon) are accreted preferentially over the unthreaded a-CD (step 26). Because the unthreaded a-CD were diluted throughout the aqueous liquid in which the PPRh was dispersed, and because the pseudorotaxanes were accreted preferentially over these unthreaded a-CD, the accreted pseudorotaxanes form a purified PPRh (pPPRh) in which unthreaded a-CD is depleted compared to in the initially formed PPRh. This accretion is typically achieved by centrifuging the aqueous liquid in which the PPRh is dispersed such that the pseudorotaxanes (i.e. the axles and the threaded a-CD threaded thereon) preferentially form a pellet of pPPRh while the unthreaded a-CD preferentially remains in the supernatant. Steps 24 and 26 may be repeated until a desired degree of unthreaded a-CD removal has been achieved. pPPRh (e.g. the liquid component thereof) may have an unthreaded a-CD concentration of less than 20 mM (e.g. less than 10 mM, e.g. less than 5 mM, e.g. less than 1 mM, e.g. substantially zero).

[0292] For some implementations, the centrifugation is carried out at at least 50 x g (e.g. at least 100 x g, such as at least 200 x g) and/or less than 500 x g - e.g. 50-500 x g, e.g. 100- 300 x g, such as at approximately 100 x g or approximately 200 x g. For some implementations, the centrifugation is carried out for at least 1 min (e.g. at least 2 min, such as at least 5 min) and/or less than 30 min - e.g. for 2-10 min, such as for approximately 5 min.

[0293] For some implementations, the pPPRh is subsequently held at a temperature of between 5 and 20 degrees C (e.g. at approximately 15 degrees C) for at least 6 hours (e.g. for approximately 12 hours) in order to facilitate further consolidation of the pPPRh.

[0294] pPPRh may behave as a solid in the absence of applied force, but one that flows as a liquid in response to sufficient force, and that returns to its solid-like state (e.g. re-sets) upon removal of the force. Similarly, discrete portions of PPRh may unify to become monolithic upon being placed together (e.g. pPPRh may be self-healing). This may be due to the uncrosslinked pseudorotaxanes forming a weak solid structure at rest, a certain amount of stress being able to overcome (e.g. break) the association between the pseudorotaxanes such that they flow with the liquid component of the PPRh, and the pseudorotaxanes being able to reform the weak solid structure upon removal of the stress. For some implementations (e.g. for some formulations), pPPRh behaves as a Bingham plastic. For some implementations (e.g. for some formulations), pPPRh behaves as a fluid gel. This characteristic may be advantageous, for example in applications in which the pPPRh is to be introduced via a nozzle (e.g. injected) and/or into a site that is irregularly shaped and/or that has crevices. In contrast, crosslinked PPRh behaves more set gelatin or agar gel which, once broken, remains so.

[0295] It is to be noted that the ability to deplete unthreaded a-CD in this manner (e.g. steps 24 and 26), without destroying the PPRh, would not have been anticipated. The existence of the PPRh depends on a-CD being threaded on the axles, which is achieved by providing excess a-CD during the initial formation of the PPRh. Indeed, existing techniques involve crosslinking the axles of the PPRh in order to obstruct the threaded a-CD from unthreading during washing. However, as described hereinabove, to retain the desired flow-and-set behavior of the hydrogel, it was desirable to identify a technique by which unthreaded a-CD could be depleted without crosslinking the axles.

[0296] At this stage the pPPRh may have a storage modulus of greater than 200 Pa and/or less than 1.2 kPa - e.g. 200-1200 Pa (e.g. 200-500 Pa, such as 250-350 Pa, such as about 300 Pa; or 0.6-1.2 kPa, e.g. 0.8-1.2 kPa, e.g. 0.9-1.1 kPa, such as approximately IkPa) - e.g. depending on centrifugation conditions. For some implementations, the elasticity (e.g. storage modulus) of the pPPRh at this stage may be at least in part dependent on the centrifugation conditions during step 26 (e.g. the G force and/or duration of centrifugation). However, it may be advantageous to select centrifugation conditions according to factors other than and/or in addition to the desired elasticity of the pPPRh. Thus, for some implementations, the elasticity (e.g. the storage modulus) of the pPPRh at this stage may be lower than that which is desired for a particular application. The desired storage modulus may be greater than 0.5 kPa (e.g. greater than 0.6 kPa, e.g. greater than 0.7 kPa, e.g. greater than 0.8 kPa) and/or less than 2 kPa (e.g. less than 1.5 kPa, such as less than 1.2 kPa) - e.g. 0.5-2 kPa (e.g. 0.5-1.5 kPa, e.g. 0.6-1.4 kPa, e.g. 0.7-1.3 kPa, e.g. 0.8-1.2 kPa, e.g. 0.9-1.1 kPa), such as approximately 1 kPa - e.g. due to similarity to that of brain tissue.

[0297] Therefore an optional step 28 may be performed in which the pPPRh is stirred while being held at an elevated temperature. It is hypothesized that the heating and stirring may provide energy that promotes the formation of crystalline domains of highly-associated pseudorotaxanes, and that this increases the storage modulus of the pPPRh. The temperature at which the pPPRh is held while stirring may be greater than 50 degrees C and/or less than 80 degrees C (e.g. 50-80 degrees C, e.g. 50-70 degrees C, e.g. 55-65 degrees C, e.g. 58-62 degrees C, such as approximately 60 degrees C).

[0298] Fig. 8 shows data from an experiment in which pPPRh was held at 60 degrees C, while stirring, for various durations. At various timepoints samples were taken and cooled to 5-20 degrees C (e.g. at approximately 15 degrees C) until rheological testing was performed. During an initial period (e.g. 5 minutes) of heating and stirring, the storage modulus decreased. It is hypothesized that this may be due to an initial release of the associations between axles (e.g. the threaded a-CD thereon) formed during accretion step 26. During a subsequent period (e.g. a subsequent 5 minutes) the storage modulus began to return toward its initial value. During a further subsequent period (e.g. between 10 and 25 minutes), a linear increase of storage modulus over time was observed. It is to be noted that this occurred despite the proportion of the solid component of the pPPRh being stable, indicating that evaporation of the liquid component was not significant and therefore did not contribute, at least not significantly, to the increase in storage modulus. It is further hypothesized that the linearity of the increase in storage modulus provides reliable control of the storage modulus during manufacturing of the pPPRh. That is, an advantage of this technique is that pPPRhs having various elastic moduli may be manufactured simply by altering the duration at which the pPPRh is held at the elevated temperature and stirred. Furthermore, this may allow a given elasticity to be achieved somewhat independently of the centrifugal conditions previously used to accrete the pPPRh. It is to be noted that, in contrast to the technique described with reference to Fig. 4, and previously-described techniques, this difference in elasticity is achieved without altering the a-CD:axle ratio of the pPPRh.

[0299] Although Fig. 8 shows data for samples taken at 5-minute intervals up to 30 minutes, additional samples were taken at 60, 90, 120, 150, 180 and 210 minutes. Further increase in storage modulus without significant evaporation was observed, at a slower rate, until 60 minutes, at which point a storage modulus of approximately 10 kPa had been achieved. Beyond 60 minutes, further increases in storage modulus became more associated with evaporative loss of the liquid component of the pPPRh. [0300] Therefore, in accordance with some implementations, an uncrosslinked PPRh (e.g. uncrosslinked pPPRh) is held and stirred at the elevated temperature for greater than 2 minutes and/or less than 60 minutes - e.g. 2-60 minutes, e.g. 2-30 minutes, e.g. 5-25 minutes (e.g. 10-25 minutes, such as 10-15 minutes or 15-20 minutes) or 2-10 minutes (e.g. 2-5 minutes or 5-10 minutes).

[0301] Reference is now made to Fig. 9, which is a schematic illustration of a technique 30 for distributing cells within a hydrogel, in accordance with some implementations. As described hereinabove, it may be desirable to include cells such as MSC within a hydrogel such as a PPRh or a pPPRh. Furthermore, it may be desirable that the cells be distributed evenly throughout the hydrogel. An existing approach to mixing cells into a medium is passing the cells and the medium through a mixing nozzle (or "mixing tip"). For example, a spiral mixing nozzle may be part of a mixing syringe that may have multiple chambers each containing a different material. For example, one chamber may contain a hydrogel and the other may contain a cell suspension. When driven through the mixing nozzle, the two become mixed before exiting the mixing nozzle.

[0302] An analysis was performed of the distribution of MSC within pPPRh after mixing via such a mixing nozzle (CELLMIXER (R), CELLINK, USA). Using confocal microscopy, fluorescence images were taken of D API- stained cells in the pPPRh, and the distribution of the cells was analyzed by determining the distance of each cell from its nearest 3 and 5 neighbors. The average distance to the nearest 3 neighbors was 132 microns, and the average distance to the nearest 5 neighbors was 166 microns.

[0303] In technique 30, a pre-distribution 32 of a suspension (or other preparation) 42 of cells (e.g. MSC) into a hydrogel (e.g. pPPRh) 40 is performed. In pre-distribution step 32, multiple deposits 44 of suspension 42 are deposited throughout the hydrogel. This may be achieved, for example, using a bioprinter. In the example shown, deposits 44 are elongate (e.g. vertical). Such elongate deposits 44 may be deposited by inserting a nozzle (e.g. needle) of a bioprinter to a given depth, and subsequently dispensing suspension 42 in conjunction with withdrawal of the needle, e.g. such that the suspension becomes disposed through the path along which the needle was disposed. Alternatively, deposits 44 may be discrete, punctate deposits. For some implementations, 50,000-200,000 (e.g. approximately 100,000) cells are deposited per 1 ml of hydrogel 40. For some implementations, 5-20 (e.g. approximately 10) microliters of suspension 42 is deposited, in deposits 44, per 1 ml of hydrogel 40. For some implementations, 50-200 (e.g. approximately 100) deposits 44 are deposited per 1 ml of hydrogel 40. For some implementations, each deposit 44 contains 500- 2000 (e.g. approximately 1000) cells. For some implementations, each deposit 44 contains 50-200 (e.g. approximately 100) nanoliters of suspension 42. Deposits 44 may be deposited in a regular or irregular array.

[0304] For some implementations, these values and parameters are determined according to the details of the particular case, e.g. via an algorithm run on a computer processor. For example, factors that may be inputs to the determination (e.g. to the algorithm) may include the desired final concentration of cells in the hydrogel, the cell type(s), the nature (e.g. viscosity and/or elasticity) of the hydrogel, the volume of the hydrogel that will receive the cells, the shape of the hydrogel (e.g. the shape of the container containing the hydrogel), and the nature of the subsequent mixing step 34.

[0305] Subsequently, the hydrogel (containing the deposits) is mixed (step 34) such that each of deposits 44 disperses within the hydrogel. Typically, this mixing is performed using a mixing nozzle such as that described hereinabove - e.g. the hydrogel containing the deposits is collected and passed through the mixing nozzle. However, for some implementations other mixing techniques, such as stirring, may be employed. In the resulting cells-in-hydrogel preparation 46, the cells are more evenly distributed compared to that achieved using a mixing nozzle alone. In analysis equivalent to that described hereinabove, the average distance to the nearest 3 neighbors was 106 microns, and the average distance to the nearest 5 neighbors was 133 microns. This significant reduction in distance suggests that technique 30 increases the homogeneity of cells throughout a hydrogel compared to using a mixing nozzle alone.

[0306] It is to be noted that technique 30 may be particularly suited to hydrogels that behave as Bingham plastics and/or fluid gels, e.g. as described hereinabove. For example, such hydrogels may be mixed without permanently breaking.

[0307] Reference is now made to Figs. 10A-C and 11A-C, which are schematic illustrations of a technique for using pPPRh, and of a hypothesized effect thereof, in accordance with some implementations. As described hereinabove, pPPRh may be particularly suitable to being introduced via a nozzle. Fig. 10A shows CNS tissue 50 having a lesion 52. Via a nozzle 56, pPPRh 54 is introduced into the lesion (Fig. 10B). For some implementations, PPPRh 54 contains MSC and/or other cells, which may have been introduced to the pPPRh using technique 30, described hereinabove. Optionally, axles at a surface of the pPPRh may then be crosslinked to form a cap 60 below which the axles remain substantially uncrosslinked (Fig. IOC). Cap 60 may advantageously seal the uncrosslinked pPPRh into the lesion, which may allow uncrosslinked pPPRh to be used in circumstances in which it would otherwise not be possible - e.g. due to the uncrosslinked pPPRh leaking. This crosslinking is typically achieved by applying energy (e.g. ultraviolet light) via an energy (e.g. light) source 58. Energy source 58 is shown as a discrete tool, but may alternatively be a component of a tool that also comprises nozzle 56. It is to be noted that, therefore, for some implementations, at least the majority of the pPPRh that is introduced into the site in the CNS remains uncrosslinked at the end of the procedure. For example, the access route via which the pPPRh is introduced into the site may be closed and/or the subject (e.g. the patient) may be discharged from the medical facility while at least the majority of the pPPRh remains uncrosslinked.

[0308] Whether for crosslinking of the entire PPRh (e.g. as for technique 10), or for the formation of cap 60, the crosslinking is typically facilitated by the axles having methacrylate termini, which have the potential to react - e.g. upon the administration of energy, such as in the form of ultraviolet light. Confocal fluoroscopic microscopy of an injury site in mouse brain tissue two weeks after introduction of uncrosslinked pPPRh has shown that the pPPRh spontaneously crosslinks in situ. It is hypothesized that this may occur as a result of reactive oxygen species (ROS), produced by inflammatory processes at the injury site, triggering the methacrylate termini to react. It is hypothesized that, in addition to the other advantages of introducing uncrosslinked pPPRh into the CNS (e.g. into the lesion), the presence of unreacted methacrylate termini may advantageously act as a buffer against ROS, potentially curbing pathological effects of the ROS. In the confocal fluoroscopic microscopy, upregulated expression of class III beta-tubulin was observed to coincide with regions of spontaneously-crosslinked pPPRh, suggesting that the spontaneous in situ crosslinking is beneficial to neuronal maturation.

[0309] An uncrosslinked hydrogel (e.g. PPRh) that is to be cross-linked either during manufacture (e.g. as per technique 10) or during administration (e.g. as per Figs. 10A-C), will typically include an initiator (e.g. a photoinitiator, such as 2,2-Dimethoxy-2- phenylacetophenone (DMPA) or Eithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)) to generate radicals that initiate the polymerization reaction. Conversely, an uncrosslinked hydrogel that is not to be cross-linked has no need for a photoinitiator. However, for an uncrosslinked PPRh that is not to be cross-linked, there would also be no need for the axles of the PPRh to have methacrylate (or other reactive) termini. Nonetheless, in light of the observations described hereinabove, a method is provided in which, in response to identifying a subject as having a pathological CNS condition, an uncrosslinked PPRh is introduced into the CNS - and the method may end (e.g. the access site may be closed and/or the subject may be discharged from the medical facility) while the PPRh remains uncrosslinked within the CNS. The axles of the uncrosslinked PPRh may have methacrylate termini but, despite this, the PPRh may not include a photoinitiator.

[0310] Based on in vitro and in vivo experiments, it is hypothesized that, over time, due to tissue growth (e.g. neuronal maturation) and/or biosorption of the pPPRh (potentially irrespectively of whether it has become crosslinked), the lesion may reduce in size and possibly even disappear. This is represented by the sequence of Figs. 11A-C. For applications in which cap 60 is formed, the cap may also become biosorbed.

[0311] It is to be noted that the technique described with reference to Figs. 10A-11C may be similarly applicable to pPPRh, PPRh and/or other hydrogels, mutatis mutandis.

[0312] In light of the above, there is provided, in accordance with some implementations, a material for introduction into a CNS site of a subject, the material comprising a PPRh that comprises alpha-cyclodextrin (a-CD) threaded onto uncrosslinked axles of a polymer that comprises ethylene oxide (e.g. the polymer is poly(ethylene oxide); PEO).

[0313] In light of the above, there is provided, in accordance with some implementations, an uncrosslinked PPRh for use as a medicament.

[0314] In light of the above, there is provided, in accordance with some implementations, an uncrosslinked PPRh for use in the treatment of a pathological CNS condition.

[0315] In light of the above, there is provided, in accordance with some implementations, an uncrosslinked PPRh for use in the treatment of a traumatic CNS injury.

[0316] In light of the above, there is provided, in accordance with some implementations, apparatus, comprising any of the above PPRhs and an applicator, configured to introduce the PPRh into a site in a central nervous system of a subject. The apparatus may be provided with the PPRh loaded in the applicator.

[0317] Example Implementations (some non-limiting examples of the concepts herein are recited below):

[0318] Example 1. A method, comprising: converting a polyp seudorotaxane hydrogel (PPRh) into a purified polypseudorotaxane hydrogel (pPPRh) by: dispersing the PPRh in an aqueous liquid, the PPRh including: axles of a polymer that includes ethylene oxide, alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and a-CD not threaded onto the axles (unthreaded a-CD); subsequently, accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD, such that the unthreaded a-CD is depleted in the pPPRh compared to in the PPRh; and using the pPPRh for manufacturing a medicament for Central Nervous System (CNS) introduction.

[0319] Example 2. The method according to example 1, wherein the pPPRh behaves as a Bingham plastic, and forming the pPPRh comprises forming the pPPRh that behaves as a Bingham plastic.

[0320] Example 3. The method according to any one of examples 1-2, wherein the medicament is for piping into the brain, and wherein using the pPPRh for manufacturing the medicament comprises using the pPPRh for manufacturing the medicament that is for piping into the brain.

[0321] Example 4. The method according to any one of examples 1-3, wherein the medicament is for treatment of traumatic brain injury, and wherein using the pPPRh for manufacturing the medicament comprises using the pPPRh for manufacturing the medicament that is for treatment of traumatic brain injury.

[0322] Example 5. The method according to any one of examples 1-4, wherein a concentration of unthreaded a-CD in the PPRh is cytotoxic, and wherein converting the PPRh into the pPPRh comprises depleting the unthreaded a-CD to a lower concentration that is less cytotoxic.

[0323] Example 6. The method according to any one of examples 1-5, wherein accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD comprises centrifuging the aqueous liquid in which the PPRh is dispersed such that the axles and the threaded a-CD preferentially accrete as a pellet while the unthreaded a-CD preferentially remains in a supernatant.

[0324] Example 7. The method according to any one of examples 1-6, wherein dispersing the PPRh in the aqueous liquid comprises dispersing the PPRh in the aqueous liquid while the aqueous liquid is at a temperature of between 50 and 70 degrees C. [0325] Example 8. The method according to any one of examples 1-7, wherein, in the pPPRh, the axles are substantially uncrosslinked.

[0326] Example 9. The method according to any one of examples 1-8, wherein the pPPRh has an storage modulus of Example 0.3-Example 1.5 kPa, and wherein forming the pPPRh comprises forming the pPPRh that has the storage modulus of Example 0.3 -Example 1.5 kPa.

[0327] Example 10. The method according to any one of examples 1-9, further comprising forming the PPRh prior to dispersing the PPRh.

[0328] Example 11. The method according to any one of examples 1-10, wherein the PPRh contains unthreaded a-CD at greater than 200 mM, the pPPRh contains unthreaded a- CD at less than 20 mM, and converting the PPRh into the pPPRh comprises converting the PPRh that contains unthreaded a-CD at greater than 200 mM into the pPPRh that contains unthreaded a-CD at less than 20 mM.

[0329] Example 12. The method according to any one of examples 1-11, wherein converting the PPRh into the pPPRh comprises depleting the unthreaded a-CD in the PPRh by at least 90%.

[0330] Example 13. The method according to any one of examples 1-12, further comprising, subsequently to forming the pPPRh: holding the pPPRh at a temperature of between 50 and 80 degrees C, and while the pPPRh is at the temperature, stirring the pPPRh.

[0331] Example 14. The method according to example 13, wherein: forming the pPPRh comprises forming the pPPRh by accreting the axles and the threaded a-CD such that the pPPRh has a first storage modulus, the method further comprises ceasing to hold and stir the pPPRh at the temperature, and holding and stirring the pPPRh comprises holding and stirring the pPPRh for a duration such that, upon ceasing to hold and stir the pPPRh, the pPPRh has a second storage modulus that is greater than the first storage modulus.

[0332] Example 15. The method according to example 14, wherein the first storage modulus is Example 0.3-1 kPa.

[0333] Example 16. The method according to example 15, wherein the second storage modulus is approximately 1 kPa. [0334] Example 17. The method according to example 13, wherein the temperature is between 50 and 70 degrees C, and holding the pPPRh at the temperature comprises holding the pPPRh at the temperature that is between 50 and 70 degrees C.

[0335] Example 18. The method according to example 13, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 2 and 10 min.

[0336] Example 19. The method according to any one of examples 1-18, further comprising dispersing cells within the pPPRh.

[0337] Example 20. The method according to example 19, wherein the cells are mesenchymal stem cells (MSC), and dispersing the cells within the pPPRh comprises dispersing the MSC within the pPPRh.

[0338] Example 21. The method according to example 19, wherein dispersing the cells within the pPPRh comprises: introducing the cells into the pPPRh; and subsequently, mixing the pPPRh containing the cells.

[0339] Example 22. The method according to example 21, wherein introducing the cells to the pPPRh comprises depositing, distributed throughout the pPPRh, multiple deposits of a suspension of the cells.

[0340] Example 23. The method according to example 22, wherein depositing the multiple deposits comprises depositing the multiple deposits using a bioprinter.

[0341] Example 24. The method according to example 21, wherein mixing the pPPRh comprises passing the pPPRh through a mixing nozzle.

[0342] Example 25. The method according to example 21, wherein mixing the pPPRh comprises stirring the pPPRh.

[0343] Example 26. A composition for introduction into a site in a central nervous system of a subject, the composition comprising a polyp seudorotaxane hydrogel (PPRh) that comprises alpha-cyclodextrin (a-CD) threaded onto uncrosslinked axles of a polymer that comprises ethylene oxide.

[0344] Example 27. The composition according to example 26, wherein the axles are axles of poly (ethylene oxide) (PEO). [0345] Example 28. The composition according to any one of examples 26-27, wherein the composition is for introduction into a central nervous system (CNS) of a subject.

[0346] Example 29. The composition according to any one of examples 26-28, wherein the composition is for treatment of a traumatic central nervous system (CNS) injury.

[0347] Example 30. The composition according to any one of examples 26-29, wherein the composition is for treatment of a central nervous system (CNS) disease.

[0348] Example 31. The composition according to any one of examples 26-30, wherein the composition behaves as a Bingham plastic.

[0349] Example 32. The composition according to any one of examples 26-30, wherein the composition behaves as a fluid gel.

[0350] Example 33. The composition according to any one of examples 26-32, wherein the PPRh does not comprise more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0351] Example 34. The composition according to example 33, wherein the PPRh does not comprise more than 10 mM unthreaded a-CD.

[0352] Example 35. The composition according to example 34, wherein the PPRh does not comprise more than 5 mM unthreaded a-CD.

[0353] Example 36. The composition according to example 35, wherein the PPRh does not comprise more than 1 mM unthreaded a-CD.

[0354] Example 37. The composition according to example 36, wherein the PPRh is substantially free of unthreaded a-CD.

[0355] Example 38. The composition according to any one of examples 26-37, wherein the axles have unreacted methacrylate termini.

[0356] Example 39. The composition according to example 38, wherein the composition does not comprise a photoinitiator.

[0357] Example 40. The composition according to any one of examples 26-39, wherein the composition contains mesenchymal stem cells (MSC) suspended therein.

[0358] Example 41. The composition according to example 40, wherein the composition contains MSC suspended therein at 50,000-200,000 cells per ml of PPRh. [0359] Example 42. The composition according to any one of examples 26-41, wherein the composition has a storage modulus of Example 0.5-Example 1.5 kPa.

[0360] Example 43. The composition according to example 42, wherein the composition has a storage modulus of Example 0.6-Example 1.4 kPa.

[0361] Example 44. The composition according to example 43, wherein the composition has a storage modulus of Example 0.7-Example 1.3 kPa.

[0362] Example 45. The composition according to example 44, wherein the composition has a storage modulus of Example 0.8-Example 1.2 kPa.

[0363] Example 46. The composition according to example 45, wherein the composition has a storage modulus of approximately 1 kPa.

[0364] Example 47. A method, comprising: identifying a subject as having a pathological central nervous system (CNS) condition; and in response to the identifying, introducing, into a site in the CNS, a polypseudorotaxane hydrogel (PPRh) that includes alpha-cyclodextrin (a-CD) threaded onto axles of a polymer that includes ethylene oxide, the axles being substantially uncrosslinked.

[0365] Example 48. The method according to example 47, wherein the PPRh behaves as a Bingham plastic, and wherein introducing the PPRh comprises introducing the PPRh that behaves as a Bingham plastic.

[0366] Example 49. The method according to example 47, wherein the PPRh behaves as a fluid gel, and wherein introducing the PPRh comprises introducing the PPRh that behaves as a fluid gel.

[0367] Example 50. The method according to any one of examples 47-49, wherein introducing the PPRh comprises piping the PPRh into the site.

[0368] Example 51. The method according to any one of examples 47-50, wherein introducing the PPRh comprises squeezing the PPRh through a nozzle into the site.

[0369] Example 52. The method according to any one of examples 47-51, wherein introducing the PPRh comprises introducing the PPRh via an access route into the subject, and wherein the method further comprises closing the access route while the axles remain substantially uncrosslinked. [0370] Example 53. The method according to any one of examples 47-52, wherein the axles have methacrylate termini, introducing the PPRh comprises introducing the PPRh via an access route into the site, and the method further comprises closing the access route while the methacrylate termini remain substantially unreacted.

[0371] Example 54. The method according to any one of examples 47-53, wherein the subject is a patient, introducing the PPRh comprises introducing the PPRh at a medical facility, and the method further comprises discharging the patient from the medical facility while the axles remain substantially uncrosslinked.

[0372] Example 55. The method according to any one of examples 47-54, wherein the axles have methacrylate termini, the subject is a patient, introducing the PPRh comprises introducing the PPRh at a medical facility, and the method further comprises discharging the patient from the medical facility while the methacrylate termini remain substantially unreacted.

[0373] Example 56. The method according to any one of examples 47-55, wherein the pathological CNS condition is a traumatic CNS injury, and wherein identifying the subject comprises identifying the subject as having the traumatic CNS injury.

[0374] Example 57. The method according to any one of examples 47-56, wherein the pathological CNS condition is a CNS disease, and wherein identifying the subject comprises identifying the subject as having the CNS disease.

[0375] Example 58. The method according to any one of examples 47-57, wherein the PPRh has an storage modulus of Example 0.5-Example 1.5 kPa, and wherein introducing the PPRh comprises introducing the PPRh that has the storage modulus of Example 0.5- Example 1.5 kPa.

[0376] Example 59. The method according to example 58, wherein the PPRh has an storage modulus of Example 0.6-Example 1.4 kPa, and wherein introducing the PPRh comprises introducing the PPRh that has the storage modulus of Example 0.6-Example 1.4 kPa.

[0377] Example 60. The method according to example 59, wherein the PPRh has an storage modulus of Example 0.7-Example 1.3 kPa, and wherein introducing the PPRh comprises introducing the PPRh that has the storage modulus of Example 0.7-Example 1.3 kPa. [0378] Example 61. The method according to example 60, wherein the PPRh has an storage modulus of Example 0.8-Example 1.2 kPa, and wherein introducing the PPRh comprises introducing the PPRh that has the storage modulus of Example 0.8-Example 1.2 kPa.

[0379] Example 62. The method according to example 61, wherein the PPRh has an storage modulus of approximately 1 kPa, and wherein introducing the PPRh comprises introducing the PPRh that has the storage modulus of approximately 1 kPa.

[0380] Example 63. The method according to any one of examples 47-62, wherein: the PPRh includes less than 20 mM a-CD that is not threaded on the axles (unthreaded a-CD), and introducing the PPRh comprises introducing the PPRh that includes less than 20 mM unthreaded a-CD.

[0381] Example 64. The method according to example 63, wherein: the PPRh includes less than 10 mM unthreaded a-CD, and introducing the PPRh comprises introducing the PPRh that includes less than 10 mM unthreaded a-CD.

[0382] Example 65. The method according to any one of examples 47-64, wherein the PPRh contains cells dispersed therewithin, and introducing the PPRh comprises introducing the PPRh that contains the cells dispersed therewithin.

[0383] Example 66. The method according to example 65, wherein the cells are mesenchymal stem cells (MSC), and introducing the PPRh comprises introducing the PPRh that contains the MSC dispersed therewithin.

[0384] Example 67. The method according to example 65, further comprising introducing the cells into the PPRh, and subsequently mixing the PPRh containing the cells.

[0385] Example 68. The method according to example 67, wherein introducing the cells into the PPRh comprises depositing, distributed throughout the PPRh, multiple deposits of a suspension of the cells.

[0386] Example 69. The method according to example 68, wherein depositing the multiple deposits comprises depositing the multiple deposits using a bioprinter.

[0387] Example 70. The method according to example 67, wherein mixing the pPPRh comprises passing the pPPRh through a mixing nozzle. [0388] Example 71. The method according to example 67, wherein mixing the pPPRh comprises stirring the pPPRh.

[0389] Example 72. The method according to any one of examples 47-71, further comprising, subsequently to introducing the PPRh, crosslinking the axles at a surface of the PPRh to form a cap beneath which the axles remaining uncrosslinked.

[0390] Example 73. The method according to example 72, wherein crosslinking the axles at the surface comprises crosslinking the axles at the surface by applying ultraviolet light to the surface.

[0391] Example 74. The method according to example 72, wherein introducing the PPRh comprises introducing the PPRh via an access route into the subject, and wherein the method further comprises closing the access route while the axles below the cap remain uncrosslinked.

[0392] Example 75. The method according to example 74, further comprising creating the access route.

[0393] Example 76. The method according to example 72, wherein the subject is a patient, introducing the PPRh comprises introducing the PPRh at a medical facility, and the method further comprises discharging the subject from the medical facility while the axles below the cap remain uncrosslinked.

[0394] Example 77. A method, comprising: dispersing, in an aqueous liquid, a polypseudorotaxane hydrogel (PPRh) that includes: axles of a polymer that includes ethylene oxide, a cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and a-CD not threaded onto the axles (unthreaded a-CD), wherein dispersing the PPRh in the aqueous liquid comprises dissolving the unthreaded a- CD in the aqueous liquid; and subsequently, by accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD, forming a purified PPRh (pPPRh) in which unthreaded a-CD is depleted compared to in the PPRh.

[0395] Example 78. The method according to example 77, wherein forming the pPPRh comprises forming the pPPRh without crosslinking the axles. [0396] Example 79. The method according to any one of examples 77-78, wherein the pPPRh is for use as a medicament.

[0397] Example 80. The method according to any one of examples 77-79, wherein the pPPRh behaves as a Bingham plastic, and forming the pPPRh comprises forming the pPPRh that behaves as a Bingham plastic.

[0398] Example 81. The method according to any one of examples 77-79, wherein the pPPRh behaves as a fluid gel, and forming the pPPRh comprises forming the pPPRh that behaves as a fluid gel.

[0399] Example 82. The method according to any one of examples 77-81, wherein dispersing the PPRh in the aqueous liquid comprises dispersing the PPRh in the aqueous liquid while the aqueous liquid is at a temperature of between 50 and 70 degrees C.

[0400] Example 83. The method according to any one of examples 77-82, wherein, in the PPRh, the axles are substantially uncrosslinked.

[0401] Example 84. The method according to any one of examples 77-83, wherein, in the pPPRh, the axles are substantially uncrosslinked.

[0402] Example 85. The method according to any one of examples 77-84, wherein the pPPRh has an storage modulus of Example 0.3-Example 1.5 kPa, and wherein forming the pPPRh comprises forming the pPPRh that has the storage modulus of Example 0.3 -Example 1.5 kPa.

[0403] Example 86. The method according to any one of examples 77-85, wherein forming the pPPRh comprises forming the pPPRh without applying ultraviolet light to the pPPRh.

[0404] Example 87. The method according to any one of examples 77-86, wherein each of the axles has methacrylate termini, and wherein forming the pPPRh comprises forming the pPPRh in which each of the axles has methacrylate termini.

[0405] Example 88. The method according to any one of examples 77-87, wherein forming the pPPRh comprises forming the pPPRh without including a photoinitiator in the pPPRh.

[0406] Example 89. The method according to any one of examples 77-88, wherein: the pPPRh includes no photoinitiator, and the method further includes introducing, into a subject, the pPPRh that includes no photoinitiator.

[0407] Example 90. The method according to any one of examples 77-89, wherein the axles are axles of polyethylene oxide (PEO), and wherein forming the pPPRh comprises forming the pPPRh in which the axles are axles of PEO.

[0408] Example 91. The method according to any one of examples 77-90, wherein the axles are axles of a block copolymer, and wherein forming the pPPRh comprises forming the pPPRh in which the axles are axles of the block copolymer.

[0409] Example 92. The method according to any one of examples 77-91, further comprising forming the PPRh prior to dispersing the PPRh.

[0410] Example 93. The method according to any one of examples 77-92, further comprising, subsequently to forming the pPPRh: holding the pPPRh at a temperature of between 50 and 80 degrees C, and while the pPPRh is at the temperature, stirring the pPPRh.

[0411] Example 94. The method according to example 93, wherein: forming the pPPRh comprises forming the pPPRh by accreting the axles and the threaded a-CD such that the pPPRh has a first storage modulus, the method further comprises ceasing to hold and stir the pPPRh at the temperature, and holding and stirring the pPPRh comprises holding and stirring the pPPRh for a duration such that, upon ceasing to hold and stir the pPPRh, the pPPRh has a second storage modulus that is greater than the first storage modulus.

[0412] Example 95. The method according to example 94, wherein the first storage modulus is Example 0.3-1 kPa.

[0413] Example 96. The method according to example 94, wherein the second storage modulus is Example 0.5-Example 1.5 kPa.

[0414] Example 97. The method according to example 96, wherein the second storage modulus is Example 0.6-Example 1.4 kPa.

[0415] Example 98. The method according to example 97, wherein the second storage modulus is Example 0.7-Example 1.3 kPa. [0416] Example 99. The method according to example 98, wherein the second storage modulus is Example 0.8-Example 1.2 kPa.

[0417] Example 100. The method according to example 99, wherein the second storage modulus is approximately 1 kPa.

[0418] Example 101. The method according to example 93, wherein the temperature is between 50 and 70 degrees C, and holding the pPPRh at the temperature comprises holding the pPPRh at the temperature that is between 50 and 70 degrees C.

[0419] Example 102. The method according to example 101, wherein the temperature is between 55 and 65 degrees C, and holding the pPPRh at the temperature comprises holding the pPPRh at the temperature that is between 55 and 65 degrees C.

[0420] Example 103. The method according to example 102, wherein the temperature is between 58 and 62 degrees C, and holding the pPPRh at the temperature comprises holding the pPPRh at the temperature that is between 58 and 62 degrees C.

[0421] Example 104. The method according to example 103, wherein the temperature is approximately 60 degrees C, and holding the pPPRh at the temperature comprises holding the pPPRh at the temperature that is approximately 60 degrees C.

[0422] Example 105. The method according to example 93, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 2 and 60 min.

[0423] Example 106. The method according to example 105, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 2 and 30 min.

[0424] Example 107. The method according to example 106, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 5 and 25 min.

[0425] Example 108. The method according to example 107, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 10 and 25 min.

[0426] Example 109. The method according to example 108, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 10 and 15 min. [0427] Example 110. The method according to example 108, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 15 and 25 min.

[0428] Example 111. The method according to example 106, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 2 and 10 min.

[0429] Example 112. The method according to example 111, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 2 and 5 min.

[0430] Example 113. The method according to example 111, wherein holding the solution at the temperature comprises holding the solution at the temperature for a duration of between 5 and 10 min.

[0431] Example 114. The method according to any one of examples 77-113, further comprising introducing the pPPRh into a central nervous system (CNS) of a subject.

[0432] Example 115. The method according to example 114, wherein introducing the pPPRh into the CNS comprises piping the pPPRh into the CNS.

[0433] Example 116. The method according to example 114, wherein introducing the pPPRh into the CNS comprises introducing the pPPRh into a brain of the subject.

[0434] Example 117. The method according to example 114, wherein introducing the pPPRh into the CNS comprises introducing the pPPRh into the CNS in response to identifying the subject as having a traumatic CNS injury.

[0435] Example 118. The method according to any one of examples 77-117, wherein accreting the axles and the threaded a-CD preferentially over the unthreaded a-CD comprises centrifuging the aqueous liquid in which the PPRh is dispersed such that the axles and the threaded a-CD preferentially form a pellet while the unthreaded a-CD preferentially remains in a supernatant.

[0436] Example 119. The method according to example 118, wherein centrifuging the aqueous liquid comprises centrifuging the aqueous liquid at 100-200 x g.

[0437] Example 120. The method according to example 118, wherein centrifuging the aqueous liquid comprises centrifuging the aqueous liquid for 2-10 min. [0438] Example 121. The method according to any one of examples 77-120, further comprising dispersing cells within the pPPRh.

[0439] Example 122. The method according to example 121, wherein the cells are mesenchymal stem cells (MSC), and dispersing the cells within the pPPRh comprises dispersing the MSC within the pPPRh.

[0440] Example 123. The method according to example 121, wherein dispersing the cells within the pPPRh comprises: introducing the cells into the pPPRh; and subsequently, mixing the pPPRh containing the cells.

[0441] Example 124. The method according to example 123, wherein introducing the cells to the pPPRh comprises depositing, distributed throughout the pPPRh, multiple deposits of a suspension of the cells.

[0442] Example 125. The method according to example 124, wherein depositing the multiple deposits comprises depositing the multiple deposits using a bioprinter.

[0443] Example 126. The method according to example 123, wherein mixing the pPPRh comprises passing the pPPRh through a mixing nozzle.

[0444] Example 127. The method according to example 123, wherein mixing the pPPRh comprises stirring the pPPRh.

[0445] Example 128. The method according to any one of examples 77-127, wherein the PPRh contains unthreaded a-CD at greater than 50 mM, and wherein dispersing the PPRh in the aqueous liquid comprises dispersing, in the aqueous liquid, the PPRh that contains unthreaded a-CD at greater than 50 mM.

[0446] Example 129. The method according to example 128, wherein the PPRh contains unthreaded a-CD at greater than 100 mM, and wherein dispersing the PPRh in the aqueous liquid comprises dispersing, in the aqueous liquid, the PPRh that contains unthreaded a-CD at greater than 100 mM.

[0447] Example 130. The method according to example 129, wherein the PPRh contains unthreaded a-CD at greater than 200 mM, and wherein dispersing the PPRh in the aqueous liquid comprises dispersing, in the aqueous liquid, the PPRh that contains unthreaded a-CD at greater than 200 mM. [0448] Example 131. The method according to any one of examples 77-130, wherein the pPPRh contains unthreaded a-CD at less than 20 mM, and wherein forming the pPPRh comprises forming the pPPRh that contains unthreaded a-CD at less than 20 mM.

[0449] Example 132. The method according to example 131, wherein the pPPRh contains unthreaded a-CD at less than 10 mM, and wherein forming the pPPRh comprises forming the pPPRh that contains unthreaded a-CD at less than 10 mM.

[0450] Example 133. The method according to example 132, wherein the pPPRh contains unthreaded a-CD at less than 5 mM, and wherein forming the pPPRh comprises forming the pPPRh that contains unthreaded a-CD at less than 5 mM.

[0451] Example 134. The method according to example 132, wherein the pPPRh contains substantially no unthreaded a-CD, and wherein forming the pPPRh comprises forming the pPPRh that contains substantially no unthreaded a-CD.

[0452] Example 135. A method, comprising: holding, at a temperature of between 50 and 70 degrees C, a polypseudorotaxane hydrogel (PPRh) that includes alpha-cyclodextrin (a-CD) threaded onto axles of a polymer that includes ethylene oxide, the axles being uncrosslinked; and while the PPRh remains at the temperature, stirring the PPRh.

[0453] Example 136. The method according to example 135, wherein holding the PPRh at the temperature comprises holding the PPRh at the temperature until the PPRh behaves as a Bingham plastic.

[0454] Example 137. The method according to example 135, wherein holding the PPRh at the temperature comprises holding the PPRh at the temperature until the PPRh behaves as a fluid gel.

[0455] Example 138. The method according to any one of examples 135-137, further comprising, prior to holding the PPRh at the temperature, forming the PPRh.

[0456] Example 139. Uncrosslinked polypseudorotaxane hydrogel (uPPRh) for use as a medicament.

[0457] Example 140. The uPPRh according to example 139, wherein the uPPRh comprises axles of poly(ethylene oxide) (PEO).

[0458] Example 141. The uPPRh according to any one of examples 139-140, wherein the uPPRh is for introduction into a central nervous system (CNS) of a subject. [0459] Example 142. The uPPRh according to any one of examples 139-141, wherein the uPPRh is for treatment of a traumatic central nervous system (CNS) injury.

[0460] Example 143. The uPPRh according to any one of examples 139-142, wherein the uPPRh is for treatment of a central nervous system (CNS) disease.

[0461] Example 144. The uPPRh according to any one of examples 139-143, wherein the uPPRh behaves as a Bingham plastic.

[0462] Example 145. The uPPRh according to any one of examples 139-143, wherein the uPPRh behaves as a fluid gel.

[0463] Example 146. The uPPRh according to any one of examples 139-145, wherein: the uPPRh comprises: axles of a polymer that includes ethylene oxide, and alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and the uPPRh does not comprise more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0464] Example 147. The uPPRh according to example 146, wherein the uPPRh does not comprise more than 10 mM unthreaded a-CD.

[0465] Example 148. The uPPRh according to example 147, wherein the uPPRh does not comprise more than 5 mM unthreaded a-CD.

[0466] Example 149. The uPPRh according to example 148, wherein the uPPRh does not comprise more than 1 mM unthreaded a-CD.

[0467] Example 150. The uPPRh according to example 149, wherein the uPPRh is substantially free of unthreaded a-CD.

[0468] Example 151. The uPPRh according to any one of examples 139-150, wherein the uPPRh comprises polymer axles that have unreacted methacrylate termini.

[0469] Example 152. The uPPRh according to example 151, wherein the uPPRh does not comprise a photoinitiator.

[0470] Example 153. The uPPRh according to any one of examples 139-152, wherein the uPPRh contains mesenchymal stem cells (MSC) suspended therein.

[0471] Example 154. The uPPRh according to example 153, wherein the uPPRh contains MSC suspended therein at 50,000-200,000 cells per ml of uPPRh. [0472] Example 155. The uPPRh according to any one of examples 139-154, wherein the uPPRh has a storage modulus of Example 0.5-Example 1.5 kPa.

[0473] Example 156. The uPPRh according to example 155, wherein the uPPRh has a storage modulus of Example 0.6-Example 1.4 kPa.

[0474] Example 157. The uPPRh according to example 156, wherein the uPPRh has a storage modulus of Example 0.7-Example 1.3 kPa.

[0475] Example 158. The uPPRh according to example 157, wherein the uPPRh has a storage modulus of Example 0.8-Example 1.2 kPa.

[0476] Example 159. The uPPRh according to example 158, wherein the uPPRh has a storage modulus of approximately 1 kPa.

[0477] Example 160. Uncrosslinked polyp seudorotaxane hydrogel (uPPRh) for use in the treatment of a pathological Central Nervous System (CNS) condition.

[0478] Example 161. The uPPRh according to example 160, wherein the uPPRh comprises axles of poly(ethylene oxide) (PEO).

[0479] Example 162. The uPPRh according to any one of examples 160-161, wherein the uPPRh is for introduction into the CNS of a subject.

[0480] Example 163. The uPPRh according to any one of examples 160-162, wherein the pathological CNS condition is a traumatic CNS injury.

[0481] Example 164. The uPPRh according to any one of examples 160-163, wherein the pathological CNS condition is a CNS disease.

[0482] Example 165. The uPPRh according to any one of examples 160-164, wherein the uPPRh behaves as a Bingham plastic.

[0483] Example 166. The uPPRh according to any one of examples 160-164, wherein the uPPRh behaves as a fluid gel.

[0484] Example 167. The uPPRh according to any one of examples 160-166, wherein: the uPPRh comprises: axles of a polymer that includes ethylene oxide, and alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and the uPPRh does not comprise more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD). [0485] Example 168. The uPPRh according to example 167, wherein the uPPRh does not comprise more than 10 mM unthreaded a-CD.

[0486] Example 169. The uPPRh according to example 168, wherein the uPPRh does not comprise more than 5 mM unthreaded a-CD.

[0487] Example 170. The uPPRh according to example 169, wherein the uPPRh does not comprise more than 1 mM unthreaded a-CD.

[0488] Example 171. The uPPRh according to example 170, wherein the uPPRh is substantially free of unthreaded a-CD.

[0489] Example 172. The uPPRh according to any one of examples 160-171, wherein the uPPRh comprises polymer axles that have unreacted methacrylate termini.

[0490] Example 173. The uPPRh according to example 172, wherein the uPPRh does not comprise a photoinitiator.

[0491] Example 174. The uPPRh according to any one of examples 160-173, wherein the uPPRh contains mesenchymal stem cells (MSC) suspended therein.

[0492] Example 175. The uPPRh according to example 174, wherein the uPPRh contains MSC suspended therein at 50,000-200,000 cells per ml of uPPRh.

[0493] Example 176. The uPPRh according to any one of examples 160-175, wherein the uPPRh has a storage modulus of Example 0.5-Example 1.5 kPa.

[0494] Example 177. The uPPRh according to example 176, wherein the uPPRh has a storage modulus of Example 0.6-Example 1.4 kPa.

[0495] Example 178. The uPPRh according to example 177, wherein the uPPRh has a storage modulus of Example 0.7-Example 1.3 kPa.

[0496] Example 179. The uPPRh according to example 178, wherein the uPPRh has a storage modulus of Example 0.8-Example 1.2 kPa.

[0497] Example 180. The uPPRh according to example 179, wherein the uPPRh has a storage modulus of approximately 1 kPa.

[0498] Example 181. Uncrosslinked polyp seudorotaxane hydrogel (uPPRh) for use in the treatment of a traumatic Central Nervous System (CNS) injury.

[0499] Example 182. The uPPRh according to example 181, wherein the uPPRh comprises axles of poly(ethylene oxide) (PEO). [0500] Example 183. The uPPRh according to any one of examples 181-182, wherein the uPPRh is for introduction into the CNS of a subject.

[0501] Example 184. The uPPRh according to any one of examples 181-183, wherein the uPPRh behaves as a Bingham plastic.

[0502] Example 185. The uPPRh according to any one of examples 181-183, wherein the uPPRh behaves as a fluid gel.

[0503] Example 186. The uPPRh according to any one of examples 181-185, wherein: the uPPRh comprises: axles of a polymer that includes ethylene oxide, and alpha-cyclodextrin (a-CD) threaded on the axles (threaded a-CD), and the uPPRh does not comprise more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0504] Example 187. The uPPRh according to example 186, wherein the uPPRh does not comprise more than 10 mM unthreaded a-CD.

[0505] Example 188. The uPPRh according to example 187, wherein the uPPRh does not comprise more than 5 mM unthreaded a-CD.

[0506] Example 189. The uPPRh according to example 188, wherein the uPPRh does not comprise more than 1 mM unthreaded a-CD.

[0507] Example 190. The uPPRh according to example 189, wherein the uPPRh is substantially free of unthreaded a-CD.

[0508] Example 191. The uPPRh according to any one of examples 181-190, wherein the uPPRh comprises polymer axles that have unreacted methacrylate termini.

[0509] Example 192. The uPPRh according to example 191, wherein the uPPRh does not comprise a photoinitiator.

[0510] Example 193. The uPPRh according to any one of examples 181-192, wherein the uPPRh contains mesenchymal stem cells (MSC) suspended therein.

[0511] Example 194. The uPPRh according to example 193, wherein the uPPRh contains MSC suspended therein at 50,000-200,000 cells per ml of uPPRh.

[0512] Example 195. The uPPRh according to any one of examples 181-194, wherein the uPPRh has a storage modulus of Example 0.5-Example 1.5 kPa. [0513] Example 196. The uPPRh according to example 195, wherein the uPPRh has a storage modulus of Example 0.6-Example 1.4 kPa.

[0514] Example 197. The uPPRh according to example 196, wherein the uPPRh has a storage modulus of Example 0.7-Example 1.3 kPa.

[0515] Example 198. The uPPRh according to example 197, wherein the uPPRh has a storage modulus of Example 0.8-Example 1.2 kPa.

[0516] Example 199. The uPPRh according to example 198, wherein the uPPRh has a storage modulus of approximately 1 kPa.

[0517] Example 200. Apparatus, comprising: a polyp seudorotaxane hydrogel (PPRh) that comprises alpha-cyclodextrin (a-CD) threaded onto uncrosslinked axles of a polymer that comprises ethylene oxide; and an applicator, configured to introduce the PPRh into a site in a central nervous system of a subject.

[0518] Example 201. The apparatus according to example 200, wherein the PPRh is disposed within the applicator.

[0519] Example 202. The apparatus according to any one of examples 200-201, wherein the PPRh behaves as a Bingham plastic.

[0520] Example 203. The apparatus according to any one of examples 200-201, wherein the PPRh behaves as a fluid gel.

[0521] Example 204. The apparatus according to any one of examples 200-203, wherein the applicator has a nozzle, and is configured to squeeze the PPRh through the nozzle and into the site.

[0522] Example 205. The apparatus according to any one of examples 200-204, further comprising a capping tool that comprises an ultraviolet (UV) light source, the tool configured to apply a dose of UV light to a surface of the PPRh at the site, the dose of UV light having a wavelength, duration, and intensity adapted to crosslink the axles at the surface to form a cap beneath which the axles remain uncrosslinked.

[0523] Example 206. The apparatus according to any one of examples 200-205, wherein the applicator comprises an ultraviolet (UV) light source, the applicator being configured to apply a dose of UV light to a surface of the PPRh at the site, the dose of UV light having a wavelength, duration, and intensity adapted to crosslink the axles at the surface to form a cap beneath which the axles remain uncrosslinked.

[0524] Example 207. The apparatus according to any one of examples 200-206, wherein the PPRh is for treatment of a traumatic central nervous system (CNS) injury.

[0525] Example 208. The apparatus according to any one of examples 200-207, wherein the PPRh is for treatment of a central nervous system (CNS) disease.

[0526] Example 209. The apparatus according to any one of examples 200-208, wherein the PPRh does not comprise more than 20 mM a-CD that is not threaded onto the axles (unthreaded a-CD).

[0527] Example 210. The apparatus according to example 209, wherein the PPRh does not comprise more than 10 mM unthreaded a-CD.

[0528] Example 211. The apparatus according to example 210, wherein the PPRh does not comprise more than 5 mM unthreaded a-CD.

[0529] Example 212. The apparatus according to example 211, wherein the PPRh does not comprise more than 1 mM unthreaded a-CD.

[0530] Example 213. The apparatus according to example 212, wherein the PPRh is substantially free of unthreaded a-CD.

[0531] Example 214. The apparatus according to any one of examples 200-213, wherein the axles have unreacted methacrylate termini.

[0532] Example 215. The apparatus according to example 214, wherein the PPRh does not comprise a photoinitiator.

[0533] Example 216. The apparatus according to any one of examples 200-215, wherein the PPRh contains mesenchymal stem cells (MSC) suspended therein.

[0534] Example 217. The apparatus according to example 216, wherein the PPRh contains MSC suspended therein at 50,000-200,000 cells per ml of PPRh.

[0535] Example 218. The apparatus according to any one of examples 200-217, wherein the PPRh has a storage modulus of Example 0.5-Example 1.5 kPa.

[0536] Example 219. The apparatus according to example 218, wherein the PPRh has a storage modulus of Example 0.6-Example 1.4 kPa. [0537] Example 220. The apparatus according to example 219, wherein the PPRh has a storage modulus of Example 0.7-Example 1.3 kPa.

[0538] Example 221. The apparatus according to example 220, wherein the PPRh has a storage modulus of Example 0.8-Example 1.2 kPa.

[0539] Example 222. The apparatus according to example 221, wherein the PPRh has a storage modulus of approximately 1 kPa.

[0540] Example 223. A method, comprising: depositing, into a hydrogel, multiple deposits of a suspension of cells such that the multiple deposits are distributed throughout the hydrogel; and subsequently, mixing the hydrogel such that each of the deposits disperses.

[0541] Example 224. The method according to example 223, wherein depositing the multiple deposits comprises depositing the multiple deposits using a bioprinter.

[0542] Example 225. The method according to any one of examples 223-224, wherein mixing the hydrogel comprises passing the hydrogel through a mixing nozzle.

[0543] Example 226. The method according to any one of examples 223-225, wherein mixing the hydrogel comprises stirring the hydrogel.

[0544] Example 227. The method according to any one of examples 223-226, wherein the hydrogel behaves as a Bingham plastic, and wherein mixing the hydrogel comprises mixing the hydrogel that behaves as a Bingham plastic.

[0545] Example 228. The method according to any one of examples 223-226, wherein the hydrogel behaves as a fluid gel, and wherein mixing the hydrogel comprises mixing the hydrogel that behaves as a fluid gel.

[0546] Example 229. The method according to any one of examples 223-228, wherein: the cells are mesenchymal stem cells (MSC), and depositing the multiple deposits comprises depositing the multiple deposits of the suspension of MSC.

[0547] Example 230. The method according to any one of examples 223-229, wherein depositing the multiple deposits comprises depositing multiple deposits that each contain 500-2000 of the cells. [0548] Example 231. The method according to any one of examples 223-230, wherein depositing the multiple deposits comprises depositing multiple deposits that each has a volume of 5-20 microliters.

[0549] Example 232. The method according to any one of examples 223-231, wherein depositing the multiple deposits comprises depositing 50-200 deposits per 1 ml of the hydrogel.

[0550] Example 233. The method according to any one of examples 223-232, wherein each of the deposits is a discrete punctate deposit, and wherein depositing the multiple deposits into the hydrogel comprises depositing the multiple discrete punctate deposits into the hydrogel.

[0551] Example 234. The method according to any one of examples 223-233, wherein each of the deposits is an elongate deposit, and wherein depositing the multiple deposits into the hydrogel comprises depositing the multiple elongate deposits into the hydrogel.

[0552] Example 235. The method according to any one of examples 223-234, further comprising, subsequently to mixing the hydrogel, introducing the hydrogel into a central nervous system (CNS) of a subject.

[0553] Example 236. The method according to example 235, wherein introducing the hydrogel into the CNS comprises piping the hydrogel into the CNS.

[0554] Example 237. The method according to example 235, wherein introducing the hydrogel into the CNS comprises introducing the hydrogel into a brain of the subject.

[0555] Example 238. The method according to example 235, wherein introducing the hydrogel into the CNS comprises introducing the hydrogel into the CNS in response to identifying the subject as having a traumatic CNS injury.

[0556] Example 239. The method according to any one of examples 223-238, wherein: the hydrogel has a storage modulus of Example 0.5-2 kPa, and depositing the multiple deposits into the hydrogel comprises depositing the multiple deposits into the hydrogel that has the storage modulus of Example 0.5-2 kPa.

[0557] Example 240. The method according to example 239, wherein: the hydrogel has an storage modulus of Example 0.5 -Example 1.5 kPa, and depositing the multiple deposits into the hydrogel comprises depositing the multiple deposits into the hydrogel that has the storage modulus of Example 0.5-Example 1.5 kPa. [0558] Example 241. The method according to example 240, wherein: the hydrogel has an storage modulus of Example 0.6 -Example 1.3 kPa, and depositing the multiple deposits into the hydrogel comprises depositing the multiple deposits into the hydrogel that has the storage modulus of Example 0.6-Example 1.3 kPa.

[0559] Example 242. The method according to example 241, wherein: the hydrogel has an storage modulus of Example 0.8-Example 1.2 kPa, and depositing the multiple deposits into the hydrogel comprises depositing the multiple deposits into the hydrogel that has the storage modulus of Example 0.8-Example 1.2 kPa.

[0560] Example 243. The method according to example 242, wherein: the hydrogel has an storage modulus of approximately 1 kPa, and depositing the multiple deposits into the hydrogel comprises depositing the multiple deposits into the hydrogel that has the storage modulus of approximately 1 kPa.

[0561] Example 244. The method according to any one of examples 223-243, wherein: the hydrogel is a polyp seudorotaxane hydrogel (PPRh) that includes alphacyclodextrin (a-CD) threaded on axles of a polymer that includes ethylene oxide, and depositing the deposits into the hydrogel comprises depositing the deposits into the PPRh.

[0562] Example 245. The method according to example 244, wherein: the axles are substantially uncrosslinked, depositing the deposits into the PPRh comprises depositing the deposits into the PPRh in which the axles are uncrosslinked, and mixing the hydrogel comprises mixing the PPRh in which the axles are substantially uncrosslinked.

[0563] Example 246. The method according to example 244, wherein: the PPRh includes less than 20 mM a-CD that is not threaded on the axles (unthreaded a-CD), and depositing the deposits into the PPRh comprises depositing the deposits into the PPRh that includes less than 20 mM unthreaded a-CD.

[0564] Example 247. The method according to example 246, wherein: the PPRh includes less than 10 mM unthreaded a-CD, and depositing the deposits into the PPRh comprises depositing the deposits into the

PPRh that includes less than 10 mM unthreaded a-CD. [0565] Example 248. The method according to example 247, wherein: the PPRh includes less than 5 mM unthreaded a-CD, and depositing the deposits into the PPRh comprises depositing the deposits into the PPRh that includes less than 5 mM unthreaded a-CD.

[0566] Any of the compositions (e.g. medicaments, PPRh, pPPRh) described herein may be sterile (e.g. may be sterilized, such as via irradiation, heat, and/or chemical means) - e.g. so as to be suitable for introduction into a subject.

[0567] The present invention is not limited to the examples that have been particularly shown and described hereinabove. Rather, the scope of the present invention includes both combinations and subcombinations of the various features described hereinabove, as well as variations and modifications thereof that are not in the prior art, which would occur to persons skilled in the art upon reading the foregoing description. Further, the treatment techniques, methods, steps, etc. described or suggested herein or references incorporated herein can be performed on a living animal or on a non-living simulation, such as on a cadaver, cadaver heart, simulator (e.g., with the body parts, tissue, etc. being simulated), etc.