Cutaneous wounding in adult humans and higher vertebrate animals results in scar formation. In contrast, both human and animal fetuses, at early gestational ages, exhibit skin wound healing without scarring. This distinction suggests that the repair of adult wounds by skin regeneration, rather than by fibrosis, may be achieved if adult wounds can be modified to mimic the healing process of fetal wounds. The development of gene therapy offers the possibility to specifically enhance or block the gene expression of cytokines and extracellular molecules, and thus convert adult wound healing into a healing process more similar to tissue regeneration. This article reviews the characteristics of fetal wound repair focusing on cytokine profiles and the inflammatory response to dermal injury. Also included are new developments in gene transfer techniques as well as their application in wound healing. Finally, the authors propose possible strategies of wound gene therapy, to reduce wound scarring and to promote tissue regeneration.

Liu Wei;Cao Yilin;Longaker Michael T.

Yonsei Medical Journal

2001

Cranial bone defect remains a major challenge to craniofacial surgeons because of limited availability of autologous bone graft to repair the defects and the donor site defects secondary to tissue harvesting. In contrast, tissue-engineering technique can generate a large bone tissue using small amount of autologous cells and therefore avoid these problems. Bone Marrow Stromal Cells (MSCs) have the potential of multi-lineage (including osteogenic) differentiation. The objective of this study was to investigate the potential of using autologous MSCs to repair cranial bone defects by a tissue-engineering approach. Autologous MSCs were isolated from eight adult sheep respectively and were in vitro expanded and induced to become osteogenic cells. Bilateral full-thickness defects (20 mm in diameter) of parietal bones were created in animals and the bone defects were either repaired with the bone implants constituted with MSCs and calcium alginate at the experimental side (n = 8) or treated with calcium alginate only without MSCs (n = 4) or left unrepaired (n = 4) at the control side. New bone tissues were observed either grossly or histologically at the defects of experimental group as early as 6 weeks post-repairing, but not in control groups. The engineered bone tissue became more mature at 18 weeks post-repairing. Three-dimensional computerized tomography (CT) scan revealed an almost complete repair of the defect of experimental group at 18 weeks. This study may provide insight for future clinical repair of cranial defect.

Shang Qingxin;Wang Zhuo;Liu Wei;Shi Yihui;Cui Lei;Cao Yilin

Journal of Craniofacial Surgery

2001

Large full-thickness defects of articular cartilage remain a major challenge to orthopedic surgeons because of unsatisfactory results of current therapy. Many methods, such as chondrectomy, drilling, cartilage scraping, arthroplasty, transplantation of chondrocytes, periosteum, perichondrium, as well as cartilage and bone, have been tried to repair articular cartilage defects. However, the results are far from satisfactory. In this study, we applied a tissue-engineering approach to the repair of articular cartilage defects of knee joints in a porcine model. Using isolated autologous chondrocytes, polyglycolic acid (PGA), and Pluronic, we have successfully in vivo-engineered hyaline cartilage and repaired articular cartilage defects. The surface of the repaired defects appeared smooth at 24 weeks postrepair. Histological examination demonstrated a typical hyaline cartilage structure with ideal interface healing between the engineered cartilage and the adjacent normal cartilage and underlying cancellous bone. In addition, glycosaminoglycan (GAG) levels in the engineered cartilage reached 80% of that found in native cartilage at 24 weeks postrepair. Biomechanical analysis at 24 weeks demonstrated that the biomechanical properties of the tissue-engineered cartilage were improved compared with those at an earlier stage. Thus, the results of this study may provide insight into the clinical repair of articular cartilage defects.

Liu Yanchun;Chen Fuguo;Liu Wei;Cui Lei;Shang Qingxin;Xia Wangyao;Wang Jian;Cui Yimin;Yang Guanghui;Liu Deli;Wu Juanjuan;Xu Rong;Buonocore Samuel D.;Cao Yilin

Tissue Engineering

2002

OBJECTIVE: To investigate the feasibility of chondrogenic phenotype differentiation of adult swine bone marrow stem cells(MSCs) in a defined medium as seeding cells in cartilage tissue engineering. METHODS: A volume of 5 ml bone marrow was aspirated from swine iliac crest and cultured in the complete medium of DMEM-LG for two weeks. The growth and ultrastructure of the cultured MSCs were observed. Immunohistochemistry and in situ hybridization were applied to detect the expression of collagen type II. RESULTS: The MSCs changed from a spindle-like fibroblastic appearance to a polygonal shape when transferred from the complete medium of DMEM-LG to a defined medium. A large amount of endoplasmic reticulum was observed in large Golgi ccmplex and mitochondria. The differentiation of MSCs toward chondrogenic phenotype was verified by the positive result of collagen type II through immunohistochemistry and in situ hybridization respectively. CONCLUSIONS: Bone marrow stem cells obtained from adult swine can differentiate to be chondrogenic phenotype when cultured in vitro. MSCs can likely be served as optimal autogenous cell source for cartilage tissue engineering.

Xia Wanyao;Shang Qingxin;Cui Lei;Xu Rong;Ding Xiaobang;Cao Yilin

Zhonghua Zheng Xing Wai Ke Za Zhi Zhonghua Zhengxing Waike Zazhi Chinese Journal of Plastic Surgery

2002

OBJECTIVE: To explore a feasible method to repair full-thickness skin defects with tissue engineered techniques. METHODS: The skin specimens were cut from the Changfeng hybrid swines' abdomen, then keratinocytes and fibroblasts were isolated and harvested by trypsin, EDTA and type II collagenase. The cells were seeded in petri dishes for primary culture. When the cells were in logarithmic growth phase, they were treated with dispase II (keratinocytes) or trypsin (fibroblasts) to separate them from the floor of the tissue culture dishes. A biodegradable material-pluronic F-127 was prefabricated and mixed with these cells, and then the cells-pluronic compounds were seeded evenly into polyglycolic acid (PGA). Tinally the constructs were replanted to autologous animals to repair full-thickness skin defects. Histological changes were observed in 1, 2, 4 and 8 weeks postsurgery. RESULTS: The cells-pluronic F-127-PGA compounds could repair autologous full-thickness skin defects. Histologically, the tissue engineered skin was similar to normal skin with stratified epidermis overlying a moderately thick collageneous dermis. CONCLUSION: Tissue engineered skin can repair autologous full-thickness skin defects with primary-cultured keratinocytes and fibroblasts as seed cells and PGA as a cell carrier.

Cao Yilin;Cai Xia;Cui Lei;Shang Qingxin;Liu Wei;Guan Wenxiang

Zhonghua Wai Ke Za Zhi Chinese Journal of Surgery

2002

Tendon defects remain a major concern in plastic surgery because of the limited availability of tendon autografts. Whereas immune rejection prohibits the use of tendon allografts, most prosthetic replacements also fail to achieve a satisfactory long-term result of tendon repair. The tissue engineering technique, however, can generate different tissues using autologous cells and thus may provide an optimal approach to address this concern. The purpose of this study was to test the feasibility of engineering tendon tissues with autologous tenocytes to bridge a tendon defect in either a tendon sheath open model or a partial open model in the hen. In a total of 40 Leghorn hens, flexor tendons were harvested from the left feet and were digested with 0.25% type II collagenase. The isolated tenocytes were expanded in vitro and mixed with unwoven polyglycolic acid fibers to form a cell-scaffold construct in the shape of a tendon. The constructs were wrapped with intestinal submucosa and then cultured in Dulbecco's Modified Eagle Medium plus 10% fetal bovine serum for 1 week before in vivo transplantation. On the feet, a defect of 3 to 4 cm was created at the second flexor digitorum profundus tendon by resecting a tendon fragment. The defects were bridged either with a cell-scaffold construct in the experimental group (n = 20) or with scaffold material alone in the control group (n = 20). Specimens were harvested at 8, 12, and 14 weeks postrepair for gross and histologic examination and for biomechanical analysis. In the experimental group, a cordlike tissue bridging the tendon defect was formed at 8 weeks postrepair. At 14 weeks, the engineered tendons resembled the natural tendons grossly in both color and texture. Histologic examination at 8 weeks showed that the neo-tendon contained abundant tenocytes and collagen; most collagen bundles were randomly arranged. The undegraded polyglycolic acid fibers surrounded by inflammatory cells were also observed. At 12 weeks, tenocytes and collagen fibers became longitudinally aligned, with good interface healing to normal tendon. At 14 weeks, the engineered tendons displayed a typical tendon structure hardly distinguishable from that of normal tendons. Biomechanical analysis demonstrated increased breaking strength of the engineered tendons with time, which reached 83 percent of normal tendon strength at 14 weeks. In the control group, polyglycolic acid constructs were mostly degraded at 8 weeks and disappeared at 14 weeks. However, the breaking strength of the scaffold materials accounted for only 9 percent of normal tendon strength. The results of this study indicated that tendon tissue could be engineered in vivo to bridge a tendon defect. The engineered tendons resembled natural tendons not only in gross appearance and histologic structure but also in biomechanical properties.

Cao Yilin;Liu Yongtao;Liu Wei;Shan Qingxin;Buonocore Samuel D.;Cui Lei

Plastic and Reconstructive Surgery

2002

OBJECTIVE: To repair critical-sized meniscal defects in an immunocompetent mammal by tissue engineering approach. METHODS: 15 45-day-old Changfeng crossbred pigs were selected as experimental animals. Autologous fibrochondrocytes were obtained from left knee menisci by modified Klagsbrun's method and were proliferated in vitro to a proper amount. A 1-cm-long full-layer defect of right medial meniscus was created anterior to the medial collateral ligament. PGA-fibrochondrocyte-Pluronic complex, fibrochondrocyte-Pluronic complex or PGA only was respectively implanted into the defects. We used intact menisci and untreated meniscal defects as controls. Samples respectively obtained in 9 w, 16 w and 25 w were appraised by general observation, histology, biochemistry and biomechanics. RESULTS: From the sights of general morphology, histological structure and Young's Modulus (59.7% of that in normal meniscus at 25 w), the PGA-cell-Pluronic complex can form the best quality tissue, which can stabilize the GAG ratio (74.5% of that in normal cartilage at 25 w) of femoral entocondyle cartilage. CONCLUSION: Autologous tissue-engineered fibrocartilage is a promising feasible method to regenerate or reconstruct menisci so as to hold back the degenerative changes of the knee.

Cui Yimin;Cao Yilin;Shang Qingxin;Cui Lei;Liu Wei

Zhonghua Yi Xue Za Zhi

2002

The fabrication and surface modification of a porous cell scaffold are very important in tissue engineering. Of most concern are high-density cell seeding, nutrient and oxygen supply, and cell affinity. In the present study, poly(L lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds with different pore structures were fabricated. An improved method based on Archimedes' Principle for measuring the porosity of scaffolds, using a density bottle, was developed. Anhydrous ammonia plasma treatment was used to modify surface properties to improve the cell affinity of the scaffolds. The results show that hydrophilicity and surface energy were improved. The polar N-containing groups and positive charged groups also were incorporated into the sample surface. A low-temperature treatment was used to maintain the plasma-modified surface properties effectively. It would do help to the further application of plasma treatment technique. Cell culture results showed that pores smaller than 160 μm are suitable for human skin fibroblast cell growth. Cell seeding efficiency was maintained at above 99%, which is better than the efficiency achieved with the common method of prewetting by ethanol. The plasma-treatment method also helped to resolve the problem of cell loss during cell seeding, and the negative effects of the ethanol trace on cell culture were avoided. The results suggest that anhydrous ammonia plasma treatment enhances the cell affinity of porous scaffolds. Mass transport issues also have been considered. © 2002 Wiley Periodicals, Inc.

Yang Jian;Shi Guixin;Bei Jianzhong;Wang Shenguo;Cao Yilin;Shang Qingxin;Yang Guanghui;Wang Wenjing

Journal of Biomedical Materials Research

2002

OBJECTIVE: To investigate the effect of tissue engineered tendon using autologous dermal fibroblasts in repairing tendon damage. METHODS: Skin tissues were resected from abdomen of 5 pigs. Fibroblasts were isolated from the skin pieces and cultured in vitro. Bundles of polyglycolic acid (PGA) were arranged in parallel and mixed with the suspension of fibroblasts to form a cell-scaffolded construct, which was further cultured in vitro for 1 week. The tendon of musculus flexor digitorum superficialis of pig's right leg was cut with a defect 3 cm long and then bridged with the cultured construct. Six weeks later, specimens of the regenerated tendon of right musculus flexor digitorum superficialis and specimens of corresponding left leg were taken for gross examination, microscopy and biomechanical analysis. RESULTS: After implantation of the fibroblast-biomaterial complex the wounds healed up and the pigs moved well. The histology of the implanted tendon was similar to that of natural tendon. The breaking strength and maximum tensile force were 173.0 +/- 18.2 N and 18.9 +/- 1.9 MPa, reaching 57.4% and 51.9% of those of normal tendon respectively. CONCLUSION: Skin fibroblasts can be used as seed cells to regenerate tendon with normal structure and function.

Chen Bing;Ding Xiaobang;Liu Fangjun;Cui Lei;Liu Wei;Shang Qingxin;Cao Yilin

Zhonghua Yi Xue Za Zhi

2002

Objective: To establish a simple and stable culture method for corneal endothelial, epithelial, and keratocyte cells. Methods: The endothelial cells from cornea on Descemet membrane were incubated for one day, then the primary cells were obtained by trypsin digestion. After scraping the superficial epithelium, cornea epithelial cells were isolated from limbal corneal tissues. Primary keratocytes were obtained by digesting the stoma using type II collagenase. Subculture of those cells were respectivly performed when they reached confluence. Results Primary endothelial cells became confluence in 4 - 5 days, and could be subcultivated to 6 - 7 passages. Limbal epithelial cells grew to be confluent in one week, but cells' vitality decreased and morphology change were seen till 3 - 4 passages. Keratocytes reached confluence in 6 - 7 days and grew rapidly after subcultivation till 10 passages. Conclusion: When optimum methods is selected to separate and culture the three kinds of corneal cells according to their characteristics, cells can be serially subcultivated and amplified.

Fu Yao;Fan Xianqun;Cao Yilin

Chinese Ophthalmic Research

2003