Recombinant Human Perlecan DV and Its LG3 Subdomain Are Neuroprotective and Acutely Functionally Restorative in Severe Experimental Ischemic Stroke.

Despite recent therapeutic advancements, ischemic stroke remains a major cause of death and disability. It has been previously demonstrated that ~ 85-kDa recombinant human perlecan domain V (rhPDV) binds to upregulated integrin receptors (α2ß1 and α5ß1) associated with neuroprotective and functional improvements in various animal models of acute ischemic stroke. Recombinant human perlecan laminin-like globular domain 3 (rhPDVLG3), a 21-kDa C-terminal subdomain of rhPDV, has been demonstrated to more avidly bind to the α2ß1 integrin receptor than its parent molecule and consequently was postulated to evoke significant neuroprotective and functional effects. To test this hypothesis, fifty male C57Bl/6 J mice studied in a t-MCAO model were randomly allocated to either rhPDV treatment, rhPDVLG3, or equivalent volume of PBS at the time of reperfusion in a study where all procedures and analyses were conducted blind to treatment. On post-MCAO day 7, 2,3,5-triphenyltetrazolium chloride staining of brain slices was used to quantify infarct volume. We observed that treatment with rhPDVLG3 reduced infarct volume by 65.6% (p = 0.0001), improved weight loss (p < 0.05), and improved functional outcome measures (p < 0.05) when compared to PBS controls, improvements which were generally greater in magnitude than those observed for 2 mg/kg of rhPDV. In addition, treatment with 6 mg/kg of rhPDVLG3 was observed to significantly reduce mortality due to stroke in one model, an outcome not previously observed for rhPDV. Our initial findings suggest that treatment with rhPDVLG3 provides significant improvement in neuroprotective and functional outcomes in experimental stroke models and that further investigation of rhPDVLG3 as a novel neuroprotective therapy for patients with stroke is warranted.

The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage.

BACKGROUND: Donor-site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Hypothesis/ PURPOSE: The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts. STUDY DESIGN: Controlled laboratory study. METHODS: Cartilage samples were from juvenile (<13 years old) and adult (>13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity. RESULTS: Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNA in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (P = .002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (P < .001). Juvenile chondrocytes did not stimulate lymphocyte proliferation. CONCLUSION: These results document a dramatic age-related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo. CLINICAL RELEVANCE: Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the fear of rejection, suggesting a new allogeneic approach to restoring articular cartilage in older individuals.

In vivo transplantation of neonatal ovine neocartilage allografts: determining the effectiveness of tissue transglutaminase.

Following transplantation of ovine neocartilage allografts, 26 sheep were divided into groups according to the following weight-bearing schedule: 8-week nonweight bearing (8NWB, n=14), and 8-week nonweight bearing+4-week weight bearing (8NWB+4WB, n=12). In addition, 7 and 6 sheep, respectively, in the 8NWB and 8NWB+4WB groups received tTG treatment after allograft transplantation, whereas the remaining 13 sheep in these groups did not receive tTG. Finally, 8 sheep served as sham-operated controls without allograft transplantation. After euthanasia, stifle joints were harvested for the analysis of gross appearance, chondrocyte viability, histology, and biomechanical testing. No significant differences were noted in macroscopic graft survival and union with host tissue in both 8NWB and 8NWB+4WB groups between the tTG treated and non-tTG treated animals. Analysis of histological scores demonstrated no significant difference between tTG and non-tTG treatments in both 8NWB and 8NWB+4WB groups. Confocal laser microscopic analysis of the explanted defects revealed 70%-100% cell viability in all treatment groups. This study shows that allogeneic chondrocytes harvested from neonatal donors provide sufficient metabolic activity to affect repair. Use of tTG to augment resorbable suture fixation of neocartilage grafts provided no advantage over suture alone in this pilot study.

Adenovirus mediated BMP-13 gene transfer induces chondrogenic differentiation of murine mesenchymal progenitor cells.

UNLABELLED: Chondrogenic/osteogenic differentiation of a mesenchymal progenitor stimulated by BMP-13 (CDMP-2) was studied. C3H10T1/2 cells were transduced by an adenoviral construct containing BMP-13 or BMP-2. BMP-13 supported chondrogenesis but not terminal differentiation, whereas BMP-2 stimulated endochondral ossification. The studies show that BMP-13 may fail to support terminal chondrocyte differentiation. INTRODUCTION: Bone morphogenetic protein (BMP)-13 is a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors. Although the biological functions of BMP-13 remain poorly understood, continued postnatal expression of BMP-13 in articular cartilage suggests that this protein may function in an autocrine/paracrine fashion to regulate growth and maintenance of articular cartilage. The purpose of this study was to elucidate the role of BMP-13 in chondrogenic differentiation. MATERIALS AND METHODS: Replication-deficient adenoviruses carrying human BMP-13 (Adv-hBMP13), bacterial beta-galactosidase (Adv-beta gal), and human BMP-2 (Adv-hBMP2) were constructed. Murine mesenchymal progenitor cells (C3H10T1/2) were transduced with these vectors, and differentiation to the chondrogenic lineage was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), biochemical, and histological analyses. RESULTS AND CONCLUSIONS: Our findings revealed that hBMP-13 transduced cells differentiated into round cells that stained with Alcian blue. Analysis of gene expression in hBMP-13-transduced cells demonstrated presence of cartilage-specific markers, absence of hypertrophic chondrocyte specific markers, and upregulation of proteoglycan biosynthesis. In particular, hBMP-13-transduced cells had significantly less and delayed expression of alkaline phosphatase activity and calcium mineral accumulation than hBMP-2-transduced cells. Except for BMPR-IB/ALK-6, expression of BMP receptors was identified constitutively in C3H10T1/2 cells and was not affected by the presence of either of the BMPs. In summary, hBMP-13, while stimulating chondrogenesis, failed to support differentiation to hypertrophic chondrocytes and endochondral ossification similar to hBMP-2. Thus, this may prove to be a useful strategy for cell-based regeneration of articular cartilage.