Brief description of the invention

Cell therapy holds the promise for a novel modality in the surgical toolkit; however, delivery of cells into damaged soft tissues constitutes a challenge. We hypothesized that growing stem cells on the surface of absorbable sutures in vitro and then implanting them via stitching a dissected soft tissue would be a suitable delivery route for cell therapy.

Fibronectin, poly-L-lysine and albumin coatings were used to increase attachment of human and rat bone marrow derived mesenchymal stem cells (BMSC) to poly-filament absorbable sutures in vitro. Fluorescence microscopy was performed to localize the cells on the suture. After 48 hours of incubation the albumin-coated sutures had the highest cell number and after 168 hours cell number reached confluency. In the in vivo experiments, a 10 mm incision was made on the triceps surae muscle of male Wistar rats and rat BMSC coated sutures were placed into the muscle. Two days after the implantation, cells were seen on the surface of the sutures as well as in the surrounding muscle tissue. Long-term results at 5 weeks showed that transplanted cells survived and the sutures were partly absorbed.

In conclusion, coating absorbable sutures with proteins, especially serum albumin, improves attachment and proliferation of cells, and only 48 hours in culture is enough to cover the sutures sufficiently. Using these stitches in vivo resulted in short and long term survival of cells. As a result, albumin-coated suture can be a vehicle for stem cell therapy in soft tissues.

Technical background of the invention

Cell therapies are already in the spectrum of possible treatment strategies in human diseases. Cell transplantation could be a promising option to increase healing potential in soft tissues, such as skeletal muscle. In our preliminary experiments, we transplanted bone marrow derived (BMSC) mesenchymal stem cells after skeletal muscle injury in rats, but we were unable to deliver the cell suspension precisely and keep it at the trauma site. We hypothesized, that cell coated sutures will create an easy to use method for cell transplantation, while delivered cells will remain at the injury site, ready to support regeneration. The first problem to be tackled is to reach sufficient adherence and coverage of cells on the sutures in vitro, then to test biocompatibility in an animal model in vivo.

Only a few scientific studies aimed to create bioactive sutures by covering them with stem cells have been conducted to date. Mazzocca and co-workers hypothesized that protein coated sutures might attract endogenous multipotent cells in vivo, which could help tendon to bone healing after a rotator cuff injury [1]. They coated non-absorbable sutures with type I collagen and investigated adhesion, proliferation and protein synthesis of human osteoblasts and tenocytes. They found that collagen coating improves the above mentioned functions in vitro. Yao and co-workers tested different adhesion proteins in order to support tendon healing with cell therapies, and concluded a that poly-L-lysine and fibronectin coating incorporates significant amount of mouse embryonic stem cells after 8 days of incubation [2]. However, 8 days in culture significantly change the characteristics of freshly isolated MSCs, therefore in further clinical use it would be better to decrease this time to 1 -2 days in order to achieve minimal manipulation of cells [3]. They also tested the bioactive suture on explanted tendon in vitro and found that adherent cells endure the mechanical stress created by suturing and remain metabolically active 96 hours after the transplantation [4].

In terms of in vivo biocompatibility of cell-coated sutures, there is only one published report. Pascual and colleagues tested cell coated sutures without protein coating in a rat colon anastomosis model. They concluded that adipose derived stem cells seeded on sutures have no effect on colon anastomosis healing [5]. Gut epithelium, however, has strong regenerative potential with high cellular turn-over, so it is possible that there is no room for improvement in this model for any therapy. Nonetheless, these early experiments highlighted the idea that using sutures for cell delivery methods is viable as long as suitable in vitro constructs are designed. The aim of the present study was twofold: 1, to design a coating and seeding protocol which achieves sufficient stem cell coverage within 1 -2 days in culture, and 2, to test the short and long term survival of the implanted cells in muscle tissue.

Detailed description of the invention

The present invention, therefore, concerns a suture coated with albumin (an albumin-coated suture) for use in introducing cells seeded thereon into a soft tissue of a patient. The suture of the invention advantageously comprises cells seeded thereon. The suture of the invention is advantageously coated with human serum albumin.

The cells seeded on the suture of the invention are advantageously non-embryonic stem cells, more advantageously mesenchymal stem cells, even more advantageously bone marrow derived mesenchymal stem cells (BMSC). The cells seeded on the suture of the invention are advantageously human cells.

The present invention further concerns a method for coating a surgical suture with albumin comprising incubating said suture in an albumin containing aqueous solution.

The invention further provides a method for seeding cells onto an albumin-coated suture comprising incubating an albumin-coated suture in a cell culture comprised in a liquid cell culture medium.

The invention further provides a method for using an albumin-coated suture for introducing cells seeded thereon into a soft tissue of a patient, said method comprising

- seeding cells to be introduced into said soft tissue of said patient on said albumin-coated suture,

- introducing said albumin-coated suture comprising said cells seeded thereon into said soft tissue of said patient.

The invention further concerns a method for introducing cells into a soft tissue of a patient comprising introducing an albumin-coated suture into said soft tissue of said patient, wherein said albumin-coated suture comprises said cells to be introduced seeded thereon.

In the methods of the invention for introducing cells into a soft tissue of a patient, said patient is advantageously a human or an animal, said cells are advantageously non-embryonic stem cells, more advantageously mesenchymal stem cells, even more advantageously bone marrow derived mesenchymal stem cells (BMSC), said cells being advantageously human cells. Examples

Animals used

Male Wistar rats weighing 250-300 g were used. Animals were maintained on lab chow and tap water ad lib with a 12 h day-night cycle in the conventional animal facility of the Institute of Human Physiology and Clinical Experimental Research, Budapest. The investigation was approved by the local Animal Research Committee according to the guidelines for animal experimentation.

Example 1

Stem cell harvest and culture

Wistar rats were terminated with urethane (5 mg/ml). Both tibia and femur were cleaned of tissue and placed in 70% ethanol. Under hood, both end of the bones were cut and the medulla was flushed out slowly with 10 ml medium (DMEM supplemented with 1 g/1 glutamine, 1 g/1 glucose, 10 % fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin) into 15 ml tubes. Bone marrow was centri- fuged at 1200 rpm for 8 minutes at RT and was placed in 100mm Petri dishes. After harvesting, BMSCs were maintained at 5% CO ₂ level and 37°C. Two days later, the culture was washed in Dulbecco's PBS and fresh medium was given every 48 hours [6-9]. After informed consent, human stem cells (HBMSC) were harvested during total hip replacement procedures. Human BMSCs were cultured similar to the previous cell type. The identity of cells was confirmed by the presence of lineage-specific cell surface markers with flow cytometry (BD ^® FacsCalibur, Becton Dickinson, NJ, USA). The majority of cells which were used for the study exhibited mesenchymal lineage specific cell surface markers (CD73, CD90, CD105, CD166) and lacked haematopoietic markers (CD34, CD45) (data not shown).

Example 2

Seeding cells on sutures

5-0 poly-filament absorbable sutures (Vicryl, Ethicon, #W9442) were used for the in vitro experiments. Albumin (Biotest Hungary Kft, #10547a96), fibronectin (Sigma, #F0895) and poly-L-lysine proteins (SIGMA #P4832) were applied onto sutures by freeze-drying. Mesenchymal stem cells were labeled with ^g/ml Hoechst 33342 (excitation 350 nm, emission 461 nm) for 30 minutes at 37°C. Two cm long freeze-dried sutures and native, uncoated sutures were put in a 24-well plate with low cell attachment surface (Costar, #3473), and 100,000 labeled cells in 1 ml culture medium were seeded onto each suture. After 48 hours, fresh medium was given to the suture -cell culture. After 24, 48 and 168 hours of incubation, fluorescent confocal microscopy (Zeiss LSM 510 MET A, Carl Zeiss, Jena, Germany) was performed to localize cells attached to the suture. Seven random regions were selected from the sutures, and nucleus-volume fraction was calculated with the Image J (National Institutes of Health, USA) analysis program as previously described [10]. Example 3

In vivo experiments

To visualize rat BMSC lipophilic membrane, dye Vybrant DiD (excitation 644 nm, emission 665 nm, Molecular Probes, #V22889) was applied. 1,700,000 labeled rat BMSCs were seeded onto 17 cm albumin-coated sutures in vitro and were incubated for 1 week as previously described.

Halothane anesthesia was maintained during the operation of male Wistar rats. After the skin incision, the triceps surae muscle was prepared by atraumatic methods. A 10 mm incision was made on the muscle with a scalpel and the albumin-coated sutures with rat BMSC were applied immediately. The longitudinal incision on the muscle was united with running technique. Following the transplantation process, the wound was closed and animals regained consciousness in their cages. Three animals were terminated at 48 hours. Approximately 1x0.5x0.2 mm muscle tissue with the bioactive suture in place was removed and native samples underwent fluorescent microscopy. After 5 weeks, another 3 animals were sacrificed, and muscle samples underwent immunohistochemistry.

Immunohistochemistry

The samples were embedded in paraffin followed by formalin fixation. After deparaffinaziation, the sections were washed three times with PBS. DNAse I (Sigma Aldrich, #DN-25) was used to DNAse digestion. After the antigen retrieval with Retrivagen A (BD Pharmingen, #550524) and washing with PBS, the sections were blocked in normal goat serum (Vector, #S-1000) for 1 hour. The primer mouse anti-BrDU antibody (BD Pharmingen, #555627) was solubilized in blocking serum and taken on the sections in a dilution of 1 :200 overnight at 4°C. After washing with PBS, Alexa Fluor 488 goat anti-mouse was used to visualize the labeling (30 min, room temperature) (Molecular Probes, #A11001). Hoechst 33342 (Molecular Probes, #H3570) was applied to the nuclei staining (10 min, room temperature). Before covering with Gel Mount (BioMeda, #M01) the sections were washed with distilled water.

Statistical analysis

Results are reported as mean ± SEM. Statistical significances were determined by one-way, two-way ANOVA and Bonferroni post-hoc tests, using Graphpad Prism statistical software. Probability values of P< 0.05, P<0.01 and P<0.001 were considered significant.

Experimental results

After 24 hours of incubation in vitro, cells were found on the surface of each suture (Fig 1.). No significant differences were observed between the different coatings. After 48 hours of incubation, the number of attached cells increased on every suture. In addition, at 48 hours of incubation, albumin-coated sutures adhered the highest number of stem cells. There was significant difference between albumin coating and native, uncoated sutures by human BMSCs in the nucleus volume fraction analysis (native 2,253±1,066 μιη ³ vs. albumin 53,204±12,949 μιη ³ volume fraction, p<0.01). Fibronectin and poly-L- lysine, however, were unable to attach significantly more cells compared to the native sutures (Fig 2). Rat BMSCs showed similar adhesion results to the previous cell type; a significantly higher adherent cell number was observed on the albumin-coated sutures after 48 hours (native vs. albumin: 13,215±4,479 μιη ³ vs. 59,182±19,722 μιη ³ volume fraction, p<0.05) (Fig 2). Incubation time was extended to 168 hours in order to monitor cell activity on the albumin-coated sutures. At 48 hours of incubation, as described previously, the used medium was removed along with the unattached cells and fresh medium was given. By the end of the 168 hours of incubation, the albumin- coated sutures were completely covered with mesenchymal stem cells. The longer incubation time allowed increased cell adhesion (Fig 3). Significant differences were found between 24 and 168 hours (24 hours vs. 168 hours: 27,531±8,822 μιη ³ vs. 15,3974±34,738) and between 48 and 168 hours (48 hours vs. 168 hours: 59,182±19,722 μιη ³ ν8. 15,3974±34,738) (Fig 3).

In the in vivo experiments, 48 hours after the operation Vibrant DiD labeled cells (red) were found on the surface of the sutures as well as in the muscle tissue next to the sutures, so the mechanical stress did not damage the cells. The cells started to detach from albumin-coated surface and start to migrate into the injured tissue (Fig. 4A). After 5 weeks in vivo incubation, BrDU labeled cells were still present in the muscle tissue (Fig. 4B).

Discussion of the advantages of the invention

In the present study we have shown that using serum albumin coating on regular absorbable sutures provides a milieu for seeded stem cells to reach a sufficient number in just 48 hours. Implantation of these cells through stitching the material into skeletal muscle was successful; in 2 days the cells were found on the suture and in the surrounding tissue, while at 5 weeks the cells migrated into deeper layers, and absorption of the suture was underway.

Paracrin factors and direct cell contact have been shown to support and save damaged tissues through cell therapy [7, 11-14]. For example, after skeletal muscle injury, satellite cells become active and through the production of myogen precursos initiate tissue regeneration [15]. Even though this is an effective cell population, experimental evidence shows that after muscle injury, and even in physiological conditions, BMSCs are contributing to skeletal muscle remodeling [16]. Moreover, in soft tissues, like skeletal muscle and tendon, the proliferation activity and cell turn-over is rather low; therefore delivering an enhanced number of pluripotent cells is a reasonable supporting mechanism.

One bottleneck in cell therapy in soft tissues is the delivery route of cells. Simple injections may be effective in generalized damage, such as in end-stage limb ischemia, however, getting cell suspensions into a posttraumatic tissue such as a torn tendon or lacerated muscle is less than optimal [17]. Experience gained from cardiac implantations shows that the optimal delivery route is far more important than previously thought, since even minor hypoxic or mechanical damage during the transplantation process can diminish the healing potential of cells. Therefore, we first optimized the in vitro process along the lines of previous studies.

In the present study, only poly-filament sutures were used to increase the adhesion surface. We observed that cells prefer to attach to the space between the woven filaments which may also be a sufficient protection from mechanical stress while suturing. We also hypothesized that coating the surgical sutures with proteins can improve cellular attachment. Coating of suture material with bioactive substrates is a way to further encourage cellular adhesion and proliferation in situ. Stitching is still the standard method for the repair of soft tissues because it is a simple application, has good mechanical properties and there is a wide range of available suture material. However, microscopic studies have shown that sutures placed through tendon tissues leads to a well-demarcated "acellular zone" around the suture which may persist up to 14 days until it is repopulated with healthy cells [18]. Therefore, augmenting mechanical repairs with cell therapy protocols can facilitate the repopulation of the acellular zone and induce repair. In order to improve not just the seeding efficiency, but also the proliferation of the cells, Dines et al. used sutures coated with growth differentiation factor-5 in tendon repair without cell implantation and showed a significantly higher tensile load and stiffness at 3 weeks [19]. In a similar study Rohrich et al. reported on covalently bound epidermal growth factor on Mersilene suture that showed increased cell proliferation along the suture in a rat flexor tendon suture model. While Hamada et al developed a monofilament nylon suture that continuously elutes basic fibroblast growth factor [20, 21]. They observed that tendons repaired with these BFGF-coated sutures increased biomechanical strength after repair for about 20 days. In an earlier study, Mazzocca et al. reported on a type I collagen coated suture, which led to a significant increase in adhesion and proliferation of human osteoblasts in vitro [1]. Another similar approach was to coat silk suture with the oligopeptid arginine-glycine-aspartic acid to stimulate tenocyte differentiation and proliferation [22] . In this study Kardestuncer and colleagues found a mild increase in tenocyte adhesion compared to tissue culture plastic. However, the relevance of this comparison is less important for an in vivo situation.

In our experiment, we tested albumin along with the above mentioned proteins and investigated human and rat BMSC adhesion. We observed that after 24, 48 hours, cell number is growing on every suture without further seeding which is a sign of proliferation. In our experiments we tested cell adhesion in shorter periods of time, because in cell therapies it will be extremely important to keep incubation time as low as possible to fulfill the "minimal manipulation" criteria for regulatory purposes. We found that after 48 hours of incubation, albumin coating attached the highest number of cells. To further increase cell number, we incubated albumin-coated sutures for 168 hours. It is important to note that the cell culture medium along with the non-adherent cells were removed and fresh medium was given after 48 hours. At 168 hours of incubation, larger adherent cell numbers were observed; meaning that cells adherent to the sutures at 48 hours were alive and proliferated. Yao and co-workers found a similar pattern of proliferation as in our study, where metabolic activity increased to a peak at day 5 and decreased afterwards [4]. This decrease in growth likely represents saturation of the suture surface area and competition of the cells for both space and media. Further incubation until 168 hours allowed longer time for proliferation; however, an earlier time point at 48 hours may be used in further human studies. We also found that human and rat cells behave the same and have the same attachment tendency, indicating that translating animal experimental findings to human protocols would be a justified approach. Albumin coating therefore provides a convenient milieu for BMSCs, which may have a significant role in protection from the environmental and mechanical stress created by the transplantation process.

In our present in vivo study, rat BMSCs were transplanted on the surface of albumin-coated sutures in order to increase the local number of multipotent cells. Since transdifferentation and cell fusion are possible mechanisms in tissue rebuilding, multipotent cells needs to be present at the trauma site and be able to migrate into the injured tissue after transplantation. For this reason, it is crucial for adherent cells not to remain attached to the surface of the suture too long. In addition, data shows that the remodeling phase of the tissue starts at around 24-48 hours after the injury [23]. In the present experiment, we investigated the location of the transplanted cells after 48 hours and found that the cells leave the surface of the suture and start to migrate into the tissue, meaning that exogenous, multipotent cells are present in the injured tissue when the regenerative processes are still ongoing. Moreover, after 5 weeks, the surviving cells were present in the host tissue showing that transplanted cells can survive after the suture had been partly absorbed.

In conclusion, our experiments showed that albumin coating is a suitable environment for BMSCs on the surface of regular suture materials. The cells only adhered to the albumin surface transitionally and become unattached after suturing, so they are able to migrate into the injured tissue readily to support the regenerative process. Therefore, stem-cell coated sutures may be a suitable new delivery method for cell therapy which is easy to introduce into current surgical practices.

References

1. Mazzocca, A.D., et al., Tendon and bone responses to a collagen-coated suture material. 2007, Elsevier, p. S222-S230.

2. Yao, J., et al., Bioactive sutures for tendon repair: assessment of a method of delivering pluripotential embryonic cells. 2008, Elsevier, p. 1558-1564.

3. Majd, H., et al., Dynamic Expansion Culture for Mesenchymal Stem Cells, Springer, p. 175.

4. Yao, J., T. Korotkova, and R.L. Smith, Viability and Proliferation of Pluripotential Cells Delivered to Tendon Repair Sites Using Bioactive Sutures— An In Vitro Study, Elsevier, p. 252-258.

5. Pascual, I., et al., Adipose derived mesenchymal stem cells in biosutures do not improve healing of experimental colonic anastomoses. 2008, Wiley Online Library, p. 1180-1184.

6. Kamila, G., et al., Characterization of bone marrow derived rat mesenchymal stem cells depending on donor age.

7. Kinnaird, T., et al., Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res, 2004. 94(5): p. 678-85.

8. Javazon, E.H., et al., Rat Marrow Stromal Cells are More Sensitive to Plating Density and Expand More Rapidly from Single Cell Derived Colonies than Human Marrow Stromal Cells. 2001, Wiley Online Library, p. 219-225.

9. Yoshimura, H., et al., Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. 2007, Springer, p. 449-462.

10. Sole, A., J. Mas, and I. Esteve, A new method based on image analysis for determining cyanobacterial biomass by CLSM in stratified benthic sediments. 2007, Elsevier, p. 669-673. Jackson, K.A., et al., Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. 2001, Am Soc Clin Investig. p. 1395-1402.

Cselenyak, A., et al., Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections, p. 29.

Sadat, S., et al., The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF. Biochemical and Biophysical Research Communications, 2007. 363(3): p. 674-679.

Deb, A., et al., Bone marrow-derived cardiomyocytes are present in adult human heart: a study of gender-mismatched bone marrow transplantation patients. 2003, Am Heart Assoc. p. 1247.

Horvathy, D., et al., Muscle regeneration is undisturbed by repeated polytraumatic injury. European Journal of Trauma and Emergency Surgery. 37(2): p. 161-167.

Palermo, A.T., et al., Bone marrow contribution to skeletal muscle: a physiological response to stress. 2005, Elsevier, p. 336-344.

Tateishi-Yuyama, E., et al., Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial*. 2002, Elsevier, p. 427-435.

Wong, J.K.F., et al., The cellular effect of a single interrupted suture on tendon. 2006, Elsevier, p. 358-367.

Dines, J.S., et al., The effect of growth differentiation factor-5-coated sutures on tendon repair in a rat model. 2007, Elsevier, p. S215-S221.

Rohrich, R.J., et al., Mersilene suture as a vehicle for delivery of growth factors in tendon repair. 1999. p. 1713.

Hamada, Y., et al., Effects of monofilament nylon coated with basic fibroblast growth factor on endogenous intrasynovial flexor tendon healing. 2006, Elsevier, p. 530-540.

Kardestuncer, T., et al., RGD-tethered silk substrate stimulates the differentiation of human tendon cells. 2006. p. 234.

Gebhard, F. and M. Huber-Lang, Polytrauma— pathophysiology and management principles. 2008, Springer, p. 825-831.