Background Art It is performed to replace the human soft tissue volume in order to augment the human soft tissues for their deficit and cosmetic purpose or for the purpose of removing skin wrinkles.

Existing methods of augmenting the human soft tissues are, first, there is a method that is to incise directly a portion which needs soft tissue augmentation and then to transplant self-skin fat tissue or fat tissue in which; second, there are methods to smash finely each human dermis, collagen of homogeneous or heterogeneous, synthesis material, self-fat tissue, then to inject it through a thick syringe needle.

But among these methods, the former method, namely, it is hardly used to incise the portion which needs soft tissue augmentation since incision scars remained extensively on both donor and recipient site, thus cosmetically is not suitable.

Among the latter methods, it is impermanent to inject one of human dermis and collagen of homogeneous or heterogeneous and synthesis material

into human body since each had been absorbed into the human body within one year, thus it has a demerit of injecting continuously, the said methods are used mainly in small augmentation but hardly used in large injection due to its too expensive material cost.

To transplant directly self-fat as an injectable material, its absorption rate is also very high. If extraction quantity is much needed, donor site of a patient who has little fat will be transformed because doctor must extract equal quantity or much more from in which. And it is difficult to find donor site to get large quantity hereby surgical operation is impossible.

Especially, a fact has been disclosed recently that to help maintain permanent volume in fat transplantation is not a fat cell but a preadipocyte, thus preadipocyte culture is an important key in treatment of the soft tissue deficit.

Disclosure of the Invention The existing methods have problems such that long incision scar on the donor site and cause of transformation on it, limitation of securing donor site, incision scar on the site of operation still remained. Therefore they are against the cosmetic purpose. And the past method is ineffective in large volume augmentation because the injectable materials had been absorbed within one year, what is more the cost of the said materials is very expensive.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide to a method of replacing the human soft tissue volume comprising steps of; in vitro culturing patient's preadipocytes in particles having a fine bubble structure which size is sufficient to pass through a syringe needle; then injecting and transplanting them as an injectable material to the human body.

Namely, in order to accomplish the object of the presentation, there is a method of replacing human soft tissue volume comprising of; culturing various

precursor cells in a material, which were extracted from the human body and then in vitro cultured, the said material is as a transplantable solid material having sponge structure and each particle size of 20-250m ; mixing them with a liquid as a culture fluid, a serum, a saline solution; injecting and transplanting them as an injectable material to the human body.

The above statement, the precursor cells can overcome the demerit of incision scar on the donor site, since they can be obtained from small amount of fat extracted from portion of axilla, circumference of umbilicus, hip with a 1cm of small incision and then cultured.

Sponge structure is very useful structure in cell culture and used in general cell culture.

In Sponge structure of solid material, each particle size is limited from 20 to 250, um to pass through the syringe needle but not to be eaten by macrophages.

There are chitosan sponge powder, polyethylene, PTFT particle (polytetrafluoethylene), PLGA particle (DL-lactic-co-glycolic acid) as an applicable material which is the said sponge structure of solid material.

These solid materials are not only three-dimensional cell culture scaffolds but also cell carriers. The culture was performed under condition of in vitro culture. The cultured cells were injected into a cell scaffold then became an injectable state. Thereafter they are mixed with the liquid such as the culture fluid, the serum, the saline solution.

Chitosan is a structure formed by repeating basic structures called N- acetylglucoseamine. Its efficacy has been disclosed such as biocompatibility, biodegradability, hemostatic activity, capability for wound healing, thereby it is used as a drugcarrier, an anti-cancer medicine, a hemostatic, a wound healing material in bio medicine.

This sponge having fine bubble structure has appropriate property as a cell scaffold due to ease to control bio-decomposition and its affinity for cell.

Besides, one of polyethylene, Medpore produced by POREX Company is

a porous polyethylene and used as a cartilage bone or a fungible for bone in the shape of board or block. It is manufactured into fine powder and can be used as a substitute for the said chitosan sponge powder.

PTFT particle (polytetrafluoroethyelene) is used mainly as a surgical artificial blood vessel. Expanded PTFT manufactured by Gore Company is composed of micro-sized fine holes. It can be used also by inserting into the body in the shape of board or block, which has soft tissue or blood vessel grow in the said fine holes. Also it is manufactured into fine powder and can be used as a substitute for the said chitosan sponge powder.

Like PLA, PLGA particle (DL-lactic-co-glycolic acid) is a mixture used as an insertion to human body. It is manufactured and also used in the shape of board having sponge structure. Also it is manufactured into fine powder and can be used as substitution for the said chitosan sponge powder.

As stated above, the said solid material which is not only a culture ground (a culture material) but also a cell carrier is transplantable material for the human body. The process of culturing in the solid sponge powder then injecting offers the most advantageous terms in point of the culture material providing short-term volume effects, culture easiness and high survival probability.

However, in view of simplification of process, a method is devised sufficiently without using the said solid scaffold as a culture ground and a carrier according to circumstances. This method is to extract preadipocyte; and to cut it finely after cell culture or to cut it finely without culture process; then to inject directly for the purpose of replacing human soft tissue.

The said method is exceptional, it makes use of transplanting in vitro cultured preadipocyte of which state is not solid particle but plane or three dimension for replacing the human soft tissue volume. It can be useful valuably to obtain rather long-term effect than short-term volume effect, which is soft tissue has increased as a result a preadipocyte specialized into a fat cell.

The present invention enables to remove limitation of extraction

quantity by in vitro culturing the preadipocytes. And also by transplanting the said preadipocytes, it enables to augment volume rather than to minimize volume decrease effect.

Furthermore, there is one object of the present invention using a particle having solid fine bubbles as a culture ground, which is to minimize other processes after culture procedures thereby it is possible to transplant immediately the said particle as an injectable material.

Best Mode for Carrying Out the Invention There are preadipocyte, chondroblast, osteoblast as human cells applicable to this invention.

Preadipocyte is known as a previous step cell of a fat cell, it becomes a fat cell through growth and specialization.

Fat cell has been exhausted in process of fat transplantation, actually survivor is a fat cell specialized from a preadipocyte, thus it is the most important cell when transplanting.

Recently, it is disclosed in case of offering appropriate circumstances to preadipocyte it can function as a mother cell specializing into a bone cell or a chondrocyte. Thereby the said preadipocyte is getting more important.

Chondroblast has a feature that it grows and specializes into chondrocyte. It has some problems as following. There are many portions which need it in human body, but its obtainable quantity is limited. And in order not to cause complication of donor site, it is required to extract only small quantity from the donor site.

In this respect, if it is possible to get much volume of cartilage after process of; obtaining small amount of chondroblast ; and culturing three- dimensionally it; then transplanting it, not only it can augment human soft tissue volume but also it can make necessary organs in human such as a cartilage of

joint which is necessary for restoration, an auricular cartilage and a nasal cartilage.

Osteoblast is a mother cell creating bone in human body. It is possible to obtain large quantity of bone by culturing and multiplying small amount of it from the donor site then injecting into a cell scaffold thereafter injecting the said scaffold into the human body.

The present invention as mentioned above is a method of transplanting the cultured cell through injection for the purpose of maintaining the volume, it can maximize cell viability by transplanting the cells with a culture ground itself.

However, in case of existing method of injecting simple cultured injectable material, its volume augmentation effect is insufficient, thus there is a problem to replace the human soft tissue volume.

Namely, it has been attempted to transplant only cultured cell through injection even except a fat cell. But in case of preadipocyte in order to be a fat cell as expanding its volume, a space in which the said preadipocyte can grow is required. Therefore the present invention provides the most effective method of volume augmentation by injecting three-dimensionally cultured cell into the human body.

Moreover the present invention offers a method as the best for the volume augmentation, that is to culture not a fat cell but a preadipocyte then to inject it into a three-dimensional space, in other words a scaffold in which it can grow and specialize, thereafter to transplant the said scaffold to human body.

This scaffold form is also obtained as result of many researches, in which sponge structure having a fine bubble structure is used. And it can supply the most proper nutrition to the cell which needs to grow and to specialize.

Generally, in case of chondroblast and osteoblast, they are used frequently in the cell culture, therein they don't need much nutrition when cultured. On the other hand, preadipocyte needs much nutrition when cultured, thus it is keenly required the said sponge structure having fine bubbles to

culture three-dimensionally.

In accordance with above constitution, the present invention provides one method as the simplest form, that is to extract preadipocyte; and to culture it or without culture process; and then to cut it finely; thereafter to transplant directly it as an injectable material for the purpose of replacing the human soft tissue volume.

In addition, the present invention also provides another novel method of replacing the human soft tissue volume, the said method comprising the stages of; using a scaffold such as chitosan sponge powder, polyethylene, PTFT particle (polytetrafluoroethyelene), PLGA particle (DL-lactic-co-glygolic acid); making form of the said scaffold a fine bubble structure which is injectable through a thin syringe needle; herein injecting distributively the cultured preadipocytes into the said scaffold; thereafter injecting an injectable material along with the said scaffold having a fine bubble structure and the said preadipocytes. Hereby one of the objects of the present invention is accomplished by injecting and transplanting not the state of solid particle but in vitro cultured preadipocytes in plane or in three-dimension for the purpose of replacing the human soft tissue volume, it is rather to attain long-term effect resulted from volume augment after preadipocytes specialized into fat cells than to attain short-term volume effect. And other object of the present invention, that is to create various cosmetic effects, also accomplished by various experiment for the volume augmentation effect and then by calculating exactly ratio of the said volume augmentation after transplanting preadipocytes or chondrocytes to the human body. Accordingly it is feasible to supplement the incision scar the biggest shortcoming in view of existing cosmetic effects, to perform easily an operation, and to achieve economical surgical operation.