OBJECTIVE: This paper aims to investigate the suitable cell density and the best formation time of tissue engineered autologous cartilage and to provide theoretical basis and parameters for clinical application. METHODS: The chondrocytes isolated from mini swines' ears were mixed with injectable biocompatible matrix (Pluronic), and the density of cell suspensions were 10, 20, 30, 40, 50, 60, 70 x 10(4)/ml. The chondrocyte-polymer constructs were subcutaneously injected into the abdomen of autologous swine. The specimens were observed grossly and histologically after 6 weeks, and investigated the suitable cell density. Then the chondrocyte-polymer constructs with suitable cell density were transplanted into the abdomen of autologous swine and evaluated grossly and histologically in 1, 3, 6, 9, 15 weeks after transplantation to investigate the best formation time of tissue engineered cartilage. RESULTS: The experiments demonstrated that the tissue engineered autologous cartilage was similar to the natural cartilage on animals with normal immune system in histological characteristics. The optimal chondrocyte density is 50 x 10(6)/ml, and the proper harvest time is the sixth week. CONCLUSION: With tissue engineering skills, we have identified the optimal chondrocyte density and the proper harvest time.

Xia W. Y.;Cao Y. L.;Shang Q. X.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi Zhongguo Xiufu Chongjian Waike Zazhi Chinese Journal of Reparative and Reconstructive Surgery

1999

Background: Auricles previously created by tissue engineering in nude mice used a biodegradable internal scaffold to maintain the desired shape of an ear. However, the biodegradable scaffold incited a compromising inflammatory response in subsequent experiments in immunocompetent animals. Objective: To test the hypothesis that tissue-engineered autologous cartilage can be bioincorporated with a nonreactive, permanent endoskeletal scaffold. Materials and Methods: Auricular elastic cartilage was harvested from Yorkshire swine. The chondrocytes were isolated and suspended into a hydrogel (Pluronic F-127) at a cell concentration of 5 x 10⁷ cells/mL. Nonbiodegradable endoskeletal scaffolds were formed with 1 of 5 polymers: (1) high-density polyethylene, (2) soft acrylic, (3) polymethylmethacrylate, (4) extrapurified Silastic, and (5) conventional Silastic. Three groups were studied: (1) a control group using only the 5 polymers, (2) the 5 polymers enveloped by Pluronic F-127 only, and (3) the implants coated with Pluronic F-127 seeded with chondrocytes. All constructs were implanted subdermally; implants containing cells were implanted into the same animal from which the cells had been islolated. The implants were harvested after 8 weeks of in vivo culture and histologically analyzed. Results: Only implants coated by hydrogel plus cells generated healthy new cartilage. With 3 polymers (high-density polyethylene, acrylic, and extrapurified Silastic), the coverage was nearly complete by elastic cartilage, with minimal fibrocartilage and minimal to no inflammatory reaction. The Food and Drug Administration - approved conventional Silastic implants resulted in fragments of fibrous tissue mixed with elastic cartilage plus evidence of chronic inflammation. The polymethylmethacrylate implant was intermediate in the amount of cartilage formed and degree of inflammation. Conclusions: This pilot technique combining tissue-engineered autologous elastic cartilage with a permanent biocompatible endoskeleton demonstrated success in limiting the inflammatory response to the scaffold, especially to high-density polyethylene, acrylic, and extrapurified Silastic. This model facilitates the potential to generate tissue of intricate shape, such as the human ear, by internal support.

Eavey Roland D.;Ar\u00e9valo-Silva Carlos A.;Cao Yilin;Vacanti Martin;Weng Yulai;Vacanti Charles A.

Archives of Otolaryngology Head and Neck Surgery

2000

Objective: To investigate the influence of growth factors on tissue-engineered pediatric human elastic cartilage relative to potential clinical application. Design: Controlled study. Subjects: Eleven children ranging in age from 5 to 15 years provided auricular elastic cartilage specimens measuring approximately 1 x 1 x 0.2 cm and weighing approximately 100 mg. Interventions: Three million chondrocytes were plated into 4 groups of Ham F-12 culture medium: group 1, Ham F-12 culture medium only; no growth factors (control group); group 2, Ham F-12 culture medium and basic fibroblast growth factor; group 3, Ham F- 12 culture medium and transforming growth actor β; and group 4, Ham F- 12 culture medium and a combination of both growth factors. At 3 weeks, the cells were harvested and mixed with a copolymer gel of polyethylene glycol and polypropylene oxide (Pluronic F-127). The cell solution was injected subcutaneously into athymic mice. The constructs were harvested at up to 22 weeks of in vivo culture and histologically analyzed. Results: The average number of cells generated in vitro was as follows: group 1, 12 million; group 2, 40 million; group 3, 7 million; and group 4, 35 million. Group 2 in vivo gross specimens were the largest and heaviest. Histologically, the control group and the basic fibroblast growth factor group (groups 1 and 2) exhibited characteristics compatible with normal auricular cartilage; groups 3 and 4 demonstrated cellular disorganization and moderate to severe fibrous tissue infiltration. Conclusions: Basic fibroblast growth factor demonstrates the greatest positive influence on the in vitro and in vivo growth of engineered pediatric human auricular cartilage. The results suggest that basic fibroblast growth factor has the potential for clinical application in which a goal will be to generate a large volume of tissue-engineered cartilage from a small donor specimen in a short period of time and of a quality similar to native human elastic cartilage.

Ar\u00e9valo-Silva Carlos A.;Cao Yilin;Vacanti Martin;Weng Yulai;Vacanti Charles A.;Eavey Roland D.

Archives of Otolaryngology Head and Neck Surgery

2000

OBJECTIVE: To investigate the formation of engineered cartilage in vivo with allogenic chondrocytes. METHODS AND MATERIALS: Joint cartilage from ewe embryos (Pregnancy: 100 days) was isolated and digested to obtain dissociated chondrocytes. Mixed chondrocytes with biomaterials (Pluronic 127) at the density 50 x 10(6)/ml, then implant the allograft complex subcutaneously. Collect the novel tissue every two weeks along for six months, weigh and stain (HE, Safranin O, Masson's trichrome) the sample tissues individually to evaluate the characteristics of novel tissue. RESULT: Novel cartilage can regenerate in allograft animals, with similar histological properties of chondrocytes, aminoglycin and collagen distribution to normal cartilage. The novel cartilage observed histologically showed apparent surrounding inflammatory cells in two and four week and the tissue of cartilage came to mature since the sixth week, with less inflammatory reaction, but not disappeared. CONCLUSION: Novel cartilage can generate in allograft animals with tissue engineering approach, with certain immunoreaction surrounding. Such reaction will be weakened gradually with time.

Xia W.;Cao Y.;Shang Q.

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

2000

Objective: To study the affinity of corneal stromal cells and PGA (polyglycolic acid), in order to fabricate a model of cells culture in a three dimensional environment. Methods: Approximately 1 million cultured corneal stromal cells were seeded onto the three-dimensional PGA fiber scaffolds. At the 4th day after cell seeding, MTT automated colormetric microassay was adopted to count the number of the cells attached to the PGA fibers. And at the 6th and 18th days, the complexes were observed by a scan electron microscopy (SEM). Result: Corneal stromal cells secreted the extracellular matrix (ECM) and multiplied shortly after they were adhered to the PGA. The cell adhersion rate was 71.40%. Conclusion: PGA can be fabricated into a suitable three-dimensional scaffold for corneal stromal cells to live in. This cell-polymer complex can be used for tissue engineered cornea research.

Hu X.;Shang Q.;Cao Y.

Chinese Ophthalmic Research

2001

Vacanti M. P.;Leonard J. L.;Dore B.;Bonassar L. J.;Cao Y.;Stachelek S. J.;Vacanti J. P.;O'Connell F.;Yu C. S.;Farwell A. P.;Vacanti C. A.

Transplantation Proceedings

2001

Elastic cartilage responds mitogenically in vitro to transforming growth factor-β (TGF-β) and basic fibroblast growth factor (basic FGF). We studied the effects of these growth factors separately or in a combination on porcine auricular chondrocytes in vitro and on the autologous elastic cartilage produced. Cells were harvested from the elastic auricular cartilage of 16- to 18-kg Yorkshire swine. Viability and quantification of the cells was determined. Cells were plated at equal concentration and studied in vitro in one of four identical media environments except for the growth factors: Group I contained Ham's F-12 with supplements but no growth factors, Group II also contained basic-FGF, Group III also contained TGF-β, and Group IV also contained a combination of both growth factors. After 3 weeks in vitro, the cells were chemically dissociated with 0.25% trypsin. Cell suspensions composed of 3 × 10⁷ cells/cc in 30% Pluronic F-127/Ham's F-12 were injected subcutaneously. Implants were harvested at 6, 8, 10, and 12 weeks of in vivo culture and then were examined with histologic stains. After 3 weeks of in vitro culture the total number of cells was as follows: Group I, 1.8 × 10⁸; Group II, 3.5 × 10⁸; Group III, 1.3 × 10⁸; Group IV, 2.5 × 10⁸. After 8 weeks of in vivo autologous implantation, the average weight (g) and volume (cm³) of each group was as follows: Group I, 0.7 g/0.15 cm³; Group II, 1.5 g/0.8 cm³; Group III, 0.6 g/0.1 cm³; Group IV, 1.2 g/0.3 cm³. Histologically, Groups I, II, and IV generated cartilage similar to native elastic cartilage, but Group III specimens demonstrated fibrous tissue ingrowth. Basic FGF produced the most positive enhancement on the quantity and quality of autologous tissue engineered elastic cartilage produced in this porcine model both in vitro and in vivo.

Ar\u00e9valo-Silva C. A.;Cao Y.;Weng Y.;Vacanti M.;Rodr\u00edguez A.;Vacanti C. A.;Eavey R. D.

Tissue Engineering

2001

Objective: To establish a method for culturing normal human oral keratinocytes. Methods: Specimens obtained from healthy humans undergoing oral surgery were dissociated into single cell suspensions by dispase and trypsin. The cells were grown in serum-free medium. Morphological characteristics were studied under light microscope and electron microscope. Cytokeratins were shown by immunohistochemistry. Results: Cells could be maintained in culture up to 4-5 passages or 30-50 days. Electron microscope revealed that there were desmosomes and tonofibrils in the oral keratinocytes. The cells showed positive staining for cytokeratin antibody. Conclusion: Human oral keratinocytes have been successfully grown in serial culture.

Zhou Zengtong;Zhou Haiwen;Shang Qingxin;Cao Yilin

Chinese Medical Journal

2001

OBJECTIVE: To investigate the proper cell density of tissue engineered autologous cartilage to indicate the clinical application. METHODS: The chondrocytes, isolated from mini swines' ears, were mixed with an injectable biocompatible matrix(Pluronic F127) to make the cell suspensions with the densities of 10,20, 30,40,50,60,70 x 10(6)/ml. The chondrocyte-polymer complex was injected into the subcutaneous tissue of the swines' abdomens. Each specimen was harvested and evaluated with body-mass, histological examination, and glycosaminoglycan content and type II collagen tests after 6 weeks in vivo. RESULTS: The histological examination had showed that the neo-cartilage was solid, homogenous cartilage when using 50 million chondrocytess/cc for 6 weeks. The samples with the 10 and 30 million chondrocytes/cc showed that the area of the cartilage was incomplete and separated by the remnant polymer. The mass of the samples was ranged from 30-110 mg after 6 weeks. The glycosaminoglycan content was lower from 5.8 to 9.0 percent, compared to the 9.2 percent of the normal auricular cartilage. Western-Blot had presented the type II collagen in all samples. CONCLUSION: This study has demonstrated that the qutologous cartilage could be generated by using tissue engineering technique, with the histological characteristics similar to natural cartilage. Fifty million chondrocytes per cc could yield the best quality cartilage in 6 weeks.

Xia W.;Cao Y.;Shang Q.

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

2001

OBJECTIVE: To develop an effective new material for repair of nerve defects. METHODS: The 4-week pre-degenerative and normal sciatic nerve segments of SD rats were extracted with the detergent lysophosphatidylcholine, which resulted in acellular nerve basal lamina tubes, called EDNG (extracted degenerative nerve grafts) and ENG (extracted normal nerve grafts) respectively. The morphology of the acellular nerve basal lamina tubes was revealed by conventional histological examinations, transmission electron microscopy and immunohistochemistry method. The basal lamina tubes, as allografts, were used to repair a 15 mm-gap of sciatic nerve in Wistar rats. In the control group nerve autografts (NAG) was applied. Evaluation included general observation, electromyographic examination, muscle measurement and histological observation of serial sections at 12 weeks after surgery. RESULTS: The EDNG had more ideal frame, better resilience than ENG. There was no any cell debris in EDNG, whereas in the inner wall of the tubes immunohistochemical analysis revealed clear positive staining and strong immunoreactivity of laminin under immunoelectron microscopy. After peripheral nerves were repaired, all the animals of the three groups had restoration of function to a certain extent. The result of the NAG group was the best, the EDNG group was the next and the ENG group was the inferior. CONCLUSION: This new material offers a possible solution to repair of a short defect of a peripheral nerve.

Dai C.;Wang W.;Cao Y.

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

2001