|ognizant Communication Corporation|
VOLUME 13, NUMBER 5, 2004
Cell Transplantation, Vol. 13, pp. 481-488, 2004
0963-6897/04 $20.00 + 00
Copyright © 2004 Cognizant Comm. Corp.
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Variation in Human Islet Viability Based on Different Membrane Integrity Stains
M. J. Barnett, D. McGhee-Wilson, A. M. J. Shapiro, and J. R. T. Lakey
Clinical Islet Program, University of Alberta, Edmonton, Canada
Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 ± 7.3% and 57.9 ± 7.2%, respectively (mean ± SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.
Key words: Viability; Islets of Langerhans; SYTO-13; Fluorescein diacetate; Transplantation
Address correspondence to Jonathan R. T. Lakey, Ph.D., Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Alberta T6G 2N8, Canada. Tel: (780) 492-3077; Fax: (780) 492-6331; E-mail: firstname.lastname@example.org
Retrovirally Transferred Genes Inhibit Apoptosis in an Insulin-Secreting Cell Line: Implications for Islet Transplantation
Elizabeth S. Fenjves,1 M. Sofia Ochoa,1 Carlota Gay-Rabinstein,1 Camillo Ricordi,1 and Michael A. Curran2
1Diabetes Research Institute, University of Miami, School
of Medicine, Miami, FL 33136
2University of California, Berkeley, Department of Molecular and Cellular Biology, Berkeley, CA 94720-3200
The transplantation of pancreatic islets for the treatment of type I diabetes is hindered by the enormous loss of cells due to early apoptotic events. Genetic engineering of islets with cytoprotective genes is an important strategy aimed to enhance the survival of these cells in the transplant setting. The present study was designed to evaluate and compare the effects of five genes on a cell line derived from insulin-producing b-cells, NIT-1. Cells were transduced using a Maloney murine leukemia virus (MLV) vector coding for yellow fluorescent protein (YFP) and for one of the following antiapoptotic genes: cFLIP, FADD-DN, BcL-2, PI-9, and ICAM-2. These genes were able to protect NIT-1 cells from cytokine-induced apoptosis to varying degrees ranging from no protection to significant protection equivalent to an optimal dose of a chemical caspase inhibitor. The data demonstrate that cFLIP, FADD-DN, and PI-9 are significantly more effective in protecting NIT-1 cells than BcL-2 and ICAM-2. Additionally, the data show that despite its weak in vitro inhibition of caspase-3, PI-9 affords significant protection against TNF-a-induced apoptosis in these cells. These genes may be ideal candidates to augment islet survival following transplantation.
Key words: Islet transplantation; NIT-1; Insulinoma cell line; Apoptosis; Gene delivery
Address correspondence to Elizabeth S. Fenjves, Diabetes Research Institute, University of Miami, School of Medicine, 1450 NW 10th. Ave. (R-134), Miami, FL 33136. Tel: (305) 243-6007; Fax: (305) 243-4404; E-mail: Efenjves@miami.edu
The Use of Multiparametric Monitoring During Islet Cell Isolation and Culture: A Potential Tool for In-Process Corrections of Critical Physiological Factors
Chris Fraker, Jorge Montelongo, Joel Szust, Aisha Khan, and Camillo Ricordi
Diabetes Research Institute, Cell Transplant Center, University of Miami School of Medicine, Miami, FL
Variables such as pH, pCO2, and pO2 have been established in the literature as critical factors that could affect the outcome of the islet cell processing and, therefore, the quality of the cells that could be transplanted. This report describes a highly accurate continuous multiparametric monitoring system and its evaluation for continuous monitoring of physiological variables during critical steps of the islet isolation procedure as well as during in vitro culture of the insulin-producing cells. Close monitoring of these variables could be of assistance to improve the outcome of islet cell processing, allowing to identify as soon as possible problems that could be corrected during the procedure, as well as during in vitro preservation, or shipment to remote sites.
Key words: Assessment; Monitoring; Islet; Isolation; Cell culture; Optical probes
Address correspondence to Camillo Ricordi, M.D., Stacy Joy Goodman Professor of Surgery and Medicine, Chief, Division of Cellular Transplantation, Scientific Director and Chief Academic Officer, Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136. Tel: (305) 243-6913; Fax: (305) 243-4404; E-mail: email@example.com
The Contribution of Chemokines and Chemokine Receptors to the Rejection of Fetal Proislet Allografts
Michelle. F. Solomon,1* William A. Kuziel,2 and Charmaine J. Simeonovic1
1Division of Immunology and Genetics, The John Curtin School
of Medical Research, The Australian National University, G.P.O. Box 334,
Canberra, A.C.T., 2601, Australia
2Section of Molecular Genetics and Microbiology The University of Texas at Austin, Austin, TX 78712
Chemokines regulate the recruitment of leukocytes to sites of inflammation and may therefore play an important role in lymphocyte trafficking between draining lymph nodes and pancreatic islet tissue allografts. The intragraft expression of a- and b-chemokine mRNA during the rejection of BALB/c proislet (fetal precursor islet tissue) allografts in CBA/H mice was assessed quantitatively and semiquantitatively by RT-PCR analyses. Allograft rejection was associated with the strongly enhanced (from day 4) and prolonged expression (up to day 10) of the a-chemokine IP-10 and enhanced intragraft mRNA expression of the b-chemokines MCP-1, MIP-1a, MIP-1b, RANTES, and eotaxin. Peak transcript expression was identified at day 4 (IP-10, MCP-1), day 5 (eotaxin), day 6 (MIP-1a, MIP-1b), and day 14 (RANTES). To examine the role of b-chemokine receptors in allograft rejection, additional allografts to CCR2-/- , CCR5-/-, and wild-type CCR+/+ mice were analyzed by histology, immunohistochemistry, and morphometry. In CCR5-/- mice, the intragraft recruitment of T cells and macrophages was slower and allograft destruction was delayed; in CCR2-/- mice, the initial entry of macrophages was retarded but graft survival was not prolonged. These findings suggest that IP-10 regulates the initial influx of T cells into proislet allografts, MCP-1/CCR2 signaling controls initial macrophage entry, and the MIP-1a, MIP-1b, and RANTES/CCR5 pathway contributes to the rejection response by subsequently amplifying the recruitment of T cell subpopulations required for graft destruction.
Key words: Chemokines; Allograft; Islet; Fetal; Rejection; mRNA
Address correspondence to Dr. Charmaine J. Simeonovic, Division of Immunology and Genetics, The John Curtin School of Medical Research, The Australian National University, G.P.O. Box 334, Canberra, A.C.T., 2601, Australia. Tel: 61-2-6125-4709; Fax: 61-2-6125-0413; E-mail: Charmaine.Simeonovic@anu.edu.au
*Present address: Department of Immunology, The Scripps Research Institute, IMM23, 10550 North Torrey Pines Rd, La Jolla, CA, 92037.
Factors Influencing Functional Survival of Microencapsulated Islet Grafts
Paul de Vos, Bart J. de Haan, Aalzen de Haan, Jacoba van Zanten, and Marijke M. Faas
Transplantation Biology and Immunoendocrinology, Department of Pathology and Laboratory Medicine, Section of Medical Biology, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands
Graft function of encapsulated islets is restricted in spite of the fact that inflammatory responses against capsules are limited to a portion less than 10%. It has been shown that dysfunction is accompanied by a gradual decrease in the glucose-induced insulin response (GIIR), a hyperproliferation of islet cells, and gradual necrosis. Also, limited survival is associated with the presence of macrophages in the overgrowth. In the present study, we investigate whether macrophages are the inducers of dysfunction of encapsulated grafts. Four weeks after successful transplantation of microencapsulated rat allografts we determined the GIIR, the rate of islet cell replication, and islet cell death. Also, we quantified the number of macrophages on the overgrown capsules. This assessment was applied to set up an in vitro coculture system of macrophages and encapsulated islets. We retrieved 93 ± 6.2% of the capsules of which 9.2 ± 0.3% was overgrown. The GIIR of the retrieved nonovergrown islets was reduced when compared with freshly encapsulated islets. The replication rate of the retrieved islet cells was eightfold higher than in the normal pancreas. Apoptosis was rarely observed but 37 ± 4% of the total islet surface was composed of necrosis. We found a mean of 1542 ± 217 macrophages per capsule. Coculture of 1500 NR8383 macrophages per encapsulated islets induced a substantial reduction in GIIR but a decrease instead of increase in replication. Necrosis was restricted to 13 ± 1.3% of the islet cells and was not increased by the presence of macrophages. Our observations indicate that we should focus on reduction of macrophage activation and on improving the nutrition of encapsulated islets to prevent islet cell death.
Key words: Islets; Macrophages; Alginate; Poly-L-lysine; Encapsulation; Immunoisolation
Address correspondence to P. De Vos, Ph.D., Transplantation Biology and Immunoendocrinology, Pathology and Laboratory Medicine, Section of Medical Biology, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. Tel: (31) (50) 3611045; Fax: (31) (50) 3619911; E-mail: P.DE.VOS@MED.RUG.NL
Comparison of Fetal Porcine Aggregates of Purified b-Cells Versus Islet-Like Cell Clusters as a Treatment of Diabetes
Jian Tu, Pauline Khoury, Lindy Williams, and Bernard E. Tuch
Diabetes Transplant Unit, The Prince of Wales Hospital and The University of New South Wales, Sydney, NSW 2031, Australia
Fetal pig islet-like cell clusters (ICCs) have the potential to reverse diabetes 1-5 months after transplantation. In a fetal ICC, however, b-cells constitute only 6-8% of the cells, in contrast to 65% in an adult pig islet. Attempts to purify fetal b-cells from cell clusters and compare their function to that of ICCs have not been shown previously. b-Cells were purified from ICCs isolated from the fetal pig pancreas. These were then aggregated and maintained in culture for 3 days. ICCs were isolated from fetal pig pancreas and allowed to round up in culture for 3 days. Transplantation of aggregates and ICCs (10,000 and 12,600, respectively) into diabetic immunoincompetent mice resulted in normoglycemia at 18 ± 2 and 8 ± 1 weeks, respectively (p = 0.0006). Removal of grafts after normalization of blood glucose levels resulted in rapid return of hyperglycemia in both groups. In conclusion, a purified population of immature b-cells can be produced from the fetal pig pancreas. The reason these cells take longer than ICCs to reverse diabetes when transplanted is postulated to be because of the relative lack of precursor cells from which b-cells differentiate. This finding may have implications for stem cell therapy, as other cell types, other than purified b-cells, may be necessary for appropriate function in vivo.
Key words: Fetal b-cells; Porcine; Purification; Transplantation; Diabetes
Address correspondence to Bernard E. Tuch, M.D., Ph.D., Diabetes Transplant Unit, The Prince of Wales Hospital, Barker Street, Randwick, NSW 2031, Australia. Tel: 61-2-9382 4814; Fax: 61-2-9382 4826; E-mail: firstname.lastname@example.org
Studies on the Differentiation of Dopaminergic Traits in Human Neural Progenitor Cells In Vitro and In Vivo
Ming Yang,1 Angela E. Donaldson,1 Cheryl E. Marshall,1 James Shen,2 and Lorraine Iacovitti1
1Farber Institute for Neurosciences, Department of Neurology,
Thomas Jefferson University Medical College, 900 Walnut Street, Philadelphia,
2ScienCell Research Laboratories, 4050 Sorrento Valley Boulevard, San Diego, CA 92121
The development of cell replacement therapies for the treatment of neurodegenerative disorders such as Parkinson's disease (PD) may depend upon the successful differentiation of human neural stem/progenitor cells into dopamine (DA) neurons. We show here that primary human neural progenitors (HNPs) can be expanded and maintained in culture both as neurospheres (NSPs) and attached monolayers where they develop into neurons and glia. When transplanted into the 6-hydroxydopamine-lesioned rat striatum, undifferentiated NSPs survive longer (60% graft survival at 8-16 weeks vs. 30% graft survival at 8-13 weeks) and migrate farther than their attached counterparts. While both NSP and attached cells continue to express neuronal traits after transplantation, the spontaneous expression of differentiated transmitter-related traits is not observed in either cell type. However, following predifferentiation in culture using a previously described cocktail of reagents, approximately 25% of HNPs can permanently express the DA enzyme tyrosine hydroxylase (TH), even following replating and removal of the DA differentiation cocktail. When these predifferentiated HNPs are transplanted into the brain, however, TH staining is not observed, either because expression is lost or TH-expressing cells preferentially die. Consistent with the latter view is a decrease in total cell survival and migration, and an enhanced glial response in these grafts. In contrast, we found that the overall survival of HNPs is improved when cells engraft near blood vessels or CSF compartments or when they are placed into an intact unlesioned brain, suggesting that there are factors, as yet unidentified, that can better support the development of engrafted HNPs.
Key words: Neural stem cells; Progenitor cells; Dopamine; Differentiation; Tyrosine hydroxylase; Transplantation; Parkinson's disease; 6-Hydroxydopmine lesion
Address correspondence to Lorraine Iacovitti, Ph.D., Associate Director, Farber Institute for Neurosciences, Thomas Jefferson University Medical College, Suite 462, Jefferson Hospital for Neuroscience, 900 Walnut Street, Philadelphia, PA 19107. Tel: (215) 955-8118; Fax: (215) 955-2993; E-mail: email@example.com
Photofabricated Gelatin-Based Nerve Conduits: Nerve Tissue Regeneration Potentials
Eduardo Gámez,1,4 Yoshinobu Goto,2 Kengo Nagata,5 Toru Iwaki,3 Tomio Sasaki,4 and Takehisa Matsuda1
Divisions of 1Biomedical Engineering, 2Clinical Neurophysiology, 3Neuropathology, and 4Neurosurgery, 5Graduate School of Medical Sciences and Division of Oral Anatomy and Cell Biology, Graduate School of Dental Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan
There is a strong demand for development of nerve guide conduit with prompt nerve regeneration potential for injury-induced nerve defect. Prior to study on nerve tissue engineering using Schwann cells or nerve stem cells, the effectiveness of photofabricated scaffolds based on photocurable gelatin was examined. This study describes the evaluation of in vivo nerve tissue regeneration potentials of three custom-designed and -fabricated prostheses (inner diameter, 1.2 mm; outer diameter, 2.4 mm; wall thickness, 0.60 mm; and length, 15 mm) made of photocured gelatin: a plain photocured gelatin tube (model I), a photocured gelatin tube packed with bioactive substances (laminin, fibronectin, and nerve growth factor) coimmobilized in a photocured gelatin rod (model II), and a photocured gelatin tube packed with bioactive substances coimmobilized in multifilament fibers (model III). These prostheses were implanted between the proximal and distal stumps 10 mm of the dissected right sciatic nerve of 70 adult male Lewis rats for up to 1 year. The highest regenerative potentials were found using the model III prosthesis, followed by the model II prosthesis. Markedly retarded neural regeneration was observed using the model I prosthesis. These were evaluated from the viewpoints of functional recovery, electrophysiological responses, and tissue morphological regeneration. The significance of the synergistic cooperative functions of multifilaments, which serve as a platform that provides contact guidance to direct longitudinal cell movement and tissue ingrowth and as a cell adhesive matrix with high surface area, and immobilized bioactive substances, which enhance nerve regeneration via biological stimulation, is discussed.
Key words: Photocurable gelatin scaffolds; Biodegradable nerve conduit; Photocured gelatin fibers; Schwann cells; Nerve tissue regeneration
Address correspondence to Takehisa Matsuda, Ph.D., Division of Biomedical Engineering, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi, Higashiku, Fukuoka 812-8562, Japan. Tel: +81-92-642-6210; Fax: +81-92-642-6212; E-mail: firstname.lastname@example.org
Efficient Early and Sustained Transduction of Human Fetal Mesencephalon Using Adeno-Associated Virus Type 2 Vectors
L. Tenenbaum,1,4 M. Peschanski,5 C. Melas,1,4 F. Rodesh,3 E. Lehtonen,1,4 A. Stathopoulos,1,4 T. Velu,4 J. Brotchi,1,2 and M. Levivier1,2
1Laboratory of Experimental Neurosurgery, 2Department
of Neurosurgery, and 3Department of Gynecology, Hôpital
Erasme, ULB, Belgium
4Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, ULB, Belgium
5INSERM U421, Hôpital Mondor, Créteil, France
The success of transplantation of human fetal mesencephalic tissue into the putamen of patients with Parkinson's disease (PD) is still limited by the poor survival of the graft. In animal models of fetal transplantation for PD, antiapoptotic agents, such as growth factors or caspase inhibitors, or agents counteracting oxidative stress enhance the survival and reinnervation potential of the graft. Genetic modification of the transplant could allow a local and continuous delivery of these factors at physiologically relevant doses. The major challenge remains the development of strategies to achieve both early and sustained gene delivery in the absence of vector-mediated toxicity. We recently reported that E14 rat fetal mesencephalon could be efficiently tranduced by adeno-associated virus type 2 (AAV2) vectors and that gene expression was maintained until at least 3 months after transplantation in the adult rat striatum. Here we report that an AAV2 vector can mediate the expression of the EGFP reporter gene under the control of a CMV promoter in organotypic cultures of freshly explanted solid fragments of human fetal mesencephalic tissue as early as 3 days to at least 6 weeks postinfection. These results suggest that AAV2 vectors could be used to genetically modify the human fetal tissue prior to transplantation to Parkinson's patients to promote graft survival and integration.
Key words: Dopaminergic grafts; Adeno-associated virus (AAV); Fetal mesencephalon; Transplantation; Parkinson's disease
Address correspondence to Liliane Tenenbaum, U.L.B. Campus Erasme, Bâtiment C, I.R.I.B.H.M., 808 Route de Lennik, B-1070 Bruxelles, Belgium. Tel: 32-2-5554095; Fax: 32-2-5554655; E-mail: email@example.com
In Vivo Evaluation of Pharmacologically Active Microcarriers Releasing Nerve Growth Factor and Conveying PC12 Cells
V. M. Tatard,1 M. C. Venier-Julienne,1 J. P. Benoit,1 P. Menei,1,2 and C. N. Montero-Menei
1INSERM U 646, Laboratoire d'Ingénierie de la vectorisation
particulaire, 10 rue André Boquel, 49100 Angers, France
2Service de Neurochirurgie, Centre Hospitalier Universitaire, 4 rue Larrey, 40033 Angers, France
Cell therapy will probably become a major therapeutic strategy in the coming years. Nevertheless, few cells survive transplantation when employed as a treatment for neuronal disorders. To address this problem, we have developed a new tool, the pharmacologically active microcarriers (PAM). PAM are biocompatible and biodegradable microparticles coated with cell adhesion molecules, conveying cells on their surface and presenting a controlled delivery of growth factor. Thus, the combined effect of growth factor and coating influences the transported cells by promoting their survival and differentiation and favoring their integration in the host tissue after their complete degradation. Furthermore, the released factor may also influence the microenvironment. In this study, we evaluated their efficacy using nerve growth factor (NGF)-releasing PAM and PC12 cells, in a Parkinson's disease paradigm. After implantation of NGF-releasing or unloaded PAM conveying PC12 cells, or PC12 cells alone, we studied cell survival, differentiation, and apoptosis, as well as behavior of the treated rats. We observed that the NGF-releasing PAM coated with two synthetic peptides (poly-D-lysine and fibronectin-like) induced PC12 cell differentiation and reduced cell death and proliferation. Moreover, the animals receiving this implant presented an improved amphetamine-induced rotational behavior. These findings indicate that PAM could be a promising strategy for cell therapy of neurological diseases and could be employed in other situations with fetal cell transplants or with stem cells.
Key words: Cell therapy; Microcarriers; Cell adhesion; Nerve growth factor; PC12 cells; Parkinson's disease
Address correspondence to C. N. Montero-Menei, INSERM U 646, 10 rue André Boquel, 49100 Angers, France. Fax: 33 2 41 73 58 53; E-mail: firstname.lastname@example.org
The Transfer of Ocular Cells Using Collagen
Chi-Kong Yeung, Sylvia Wai-Yee Chiang, Kwok-Ping Chan, Dennis Shun-Chiu Lam, and Chi-Pui Pang
Department of Ophthalmology & Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
The present study was designed to evaluate the use of collagen gel loaded with human retinal pigment epithelium (ARPE19) in cellular transfer and to assess its viability within the gel. Collagen solution was prepared by dissolving calfskin in hydrochloric acid to make a final concentration of 2.0 mg/ml and this was mixed with 10,000 ARPE19 cells/ml. The cell viability in gel was determined using MTT assay. van Gieson stain and proliferating cell nuclear antigen (PCNA) were used to identify the location of collagen and to localize the site of cell proliferation, respectively. The ARPE19 cells in gel appeared to be healthy with a rounded morphology. The optimal collagen concentration was 1.9 mg/ml. When this concentration was used to hold cells for over 12 days, it could be seen that the growth rate was the same between day 2 and day 8 in gel and on plastic. When the cell-loaded gels were transferred onto standard tissue culture plastics, progressive cell migrations over time resembling cell migrations in organotypic explant cultures were observed. Upon intravitreal injection of cell-containing collagen suspension into a rabbit's eye, the gel became suspended within the vitreous a few hours after injection (day 0). However, it became obvious that the gel dispersed and spread around the vitreous even after just 24 h. These cells inside the vitreous were PCNA positive, indicating that the human ARPE19 cells have the capacity to proliferate even after 11 days. The present study demonstrated the potential use of collagen gel as a tool in the transfer of cellular matrix onto other substrates. The results show that the cell seeding number must be critically balanced with the concentration of gel for it to be used as transplant material.
Key words: Cell transfer; Cellular matrix; Collagen gel; Cytotoxicity; Retinal pigment epithelium; Intravitreal injection
Address correspondence to Dr. Chi-Kong Yeung at his present address: Bioengineering Graduate Program, Department of Chemical Engineering, Room 2004A, Academic Building, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Tel: +825 2358 8433; Fax: +825 3106 4857; E-mail: email@example.com
Autologous Bone Marrow Stromal Cell Transplantation for Repair of Full-Thickness Articular Cartilage Defects in Human Patellae: Two Case Reports
Shigeyuki Wakitani,1 Tomoki Mitsuoka,2 Norimasa Nakamura,3 Yukiyoshi Toritsuka,3 Yukio Nakamura,1 and Shuji Horibe2
1Department of Orthopaedic Surgery, Shinshu University School
of Medicine, Matsumoto 390-8621, Japan
2Department of Orthopaedic Surgery, Osaka Rosai Hospital, Sakai 591-8025, Japan
3Department of Orthopaedic Surgery, Osaka University Medical School, Suita 565-0871, Japan
This study assessed the effectiveness of autologous bone marrow stromal cell transplantation for the repair of full-thickness articular cartilage defects in the patellae of a 26-year-old female and a 44-year-old male. These two patients presented in our clinic because their knee pain prevented them from walking normally. After thorough examination, we concluded that the knee pain was due to the injured articular cartilage and decided to repair the defect with bone marrow stromal cell transplantation. Three weeks before transplantation, bone marrow was aspirated from the iliac crest of each patient. After erythrocytes had been removed by use of dextran, the remaining nucleated cells were placed in culture. When the attached cells reached subconfluence, they were passaged to expand in culture. Adherent cells were subsequently collected, embedded in a collagen gel, transplanted into the articular cartilage defect in the patellae, and covered with autologous periosteum. Six months after transplantation, clinical symptoms (pain and walking ability) had improved significantly and the improvement has remained in effect (5 years and 9 months posttransplantation in one case, and 4 years in the other), and both patients have been satisfied with the outcome. As early as 2 months after transplantation, the defects were covered with tissue that showed slight metachromatic staining. Two years after the first and 1 year after the second transplantation, arthroscopy was performed and the defects were repaired with fibrocartilage. Results indicate autologous bone marrow stromal cell transplantation is an effective approach in promoting the repair of articular cartilage defects.
Key words: Chondro-progenitor cell; Cartilage regeneration; Resurfacing; Fibrocartilage
Address correspondence to Shigeyuki Wakitani, M.D., Department of Orthopaedic Surgery, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, 390-8621, Japan. Tel: +81-263-37-2659; Fax: +81-263-35-8844; E-mail: firstname.lastname@example.org