Enhanced tissue regeneration potential of juvenile articular cartilage.

BACKGROUND: Articular cartilage undergoes substantial age-related changes in molecular composition, matrix structure, and mechanical properties. These age-related differences between juvenile and adult cartilage manifest themselves as markedly distinct potentials for tissue repair and regeneration. PURPOSE: To compare the biological properties and tissue regeneration capabilities of juvenile and adult bovine articular cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage harvested from juvenile (age, 4 months) and adult (age, 6-8 years) bovine femoral condyles was cultured for 4 weeks to monitor chondrocyte migration, glycosaminoglycan content conservation, and new tissue formation. The cartilage cell density and proliferative activity were also compared. Additionally, the effects of age-related changes on cartilage gene expression were analyzed using the Affymetrix GeneChip array. RESULTS: Compared with adult cartilage, juvenile bovine cartilage demonstrated a significantly greater cell density, higher cell proliferation rate, increased cell outgrowth, elevated glycosaminoglycan content, and enhanced matrix metallopeptidase 2 activity. During 4 weeks in culture, only juvenile cartilage was able to generate new cartilaginous tissues, which exhibited pronounced labeling for proteoglycan and type II collagen but not type I collagen. With over 19,000 genes analyzed, distinctive gene expression profiles were identified. The genes mostly involved in cartilage growth and expansion, such as COL2A1, COL9A1, MMP2, MMP14, and TGFB3, were upregulated in juvenile cartilage, whereas the genes primarily responsible for structural integrity, such as COMP, FN1, TIMP2, TIMP3, and BMP2, were upregulated in adult cartilage. CONCLUSION: As the first comprehensive comparison between juvenile and adult bovine articular cartilage at the tissue, cellular, and molecular levels, the results strongly suggest that juvenile cartilage possesses superior chondrogenic activity and enhanced regenerative potential over its adult counterpart. Additionally, the differential gene expression profiles of juvenile and adult cartilage suggest possible mechanisms underlying cartilage age-related changes in their regeneration capabilities, structural components, and biological properties. CLINICAL RELEVANCE: The results of this comparative study between juvenile and adult bovine articular cartilage suggest an enhanced regenerative potential of juvenile cartilage tissue in the restoration of damaged articular cartilage.

Transdermal lovastatin enhances fracture repair in rats.

Statins have been shown to stimulate BMP2 transcription and bone formation. This raises the possibility that they could be useful for enhancing rates of fracture repair. Observational studies in patients treated with oral statins for lipid-lowering have been controversial. The likely reason for their inconsistent effects is that the statin concentration reaching the periphery was too low after oral administration to produce a reproducible biologic effect. Thus, we examined the effects of lovastatin (LV) given transdermally in a well-described preclinical model of fracture repair. Effects on the healing fracture callus were assessed by biomechanical strength, radiographs, and quantitative morphology. LV was administered transdermally (TD) for 5 days after fracture in several doses (0.1-5 mg/kg/d) and compared with vehicle-treated control rats and rats treated with LV by oral gavage (PO) at 5-25 mg/kg/d for 5 days from the day of fracture. Radiological evaluation of bones treated with TD LV showed enhanced fracture repair at 2 and 6 wk. BMD in the callus area at 6 wk was also increased in the TD group compared with vehicle-treated controls (p < 0.05). The force required to break TD-treated bones (0.1 mg/kg/d for 5 days) was 42% greater than vehicle-treated controls (p < 0.02), and there was a 90% increase in stiffness (p < 0.01). PO LV at much higher doses (10 and 25 mg/kg/d) showed increased stiffness but no change in other biomechanical properties. By histological examination, a significant increase was also observed in the size of the callus, surrounding proliferating cell nuclear antigen-positive cells, and osteoblast and osteoclast number in TD-treated rats compared with controls at day 8 after fracture (n = 6). In summary, we found that TD LV in low doses accelerates fracture healing, whereas 10-fold the lipid-lowering dose was required to produce any effect when it was administered orally. These studies provide valuable information on the potential of statins and TD delivery as a new and effective therapeutic modality in fracture repair.

Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE-ASARM motif: a model for impaired mineralization in X-linked rickets (HYP).

Matrix Extracellular Phospho-glycoprotEin (MEPE) and proteases are elevated and PHEX is defective in HYP. PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE-ASARM peptide, an inhibitor of mineralization (minhibin). Thus, in HYP, mutated PHEX may contribute to increased ASARM peptide release. Moreover, binding of MEPE by PHEX may regulate this process in normal subjects. The nature of the PHEX-MEPE nonproteolytic interaction(s) (direct or indirect) is/are unknown. Our aims were to determine (1) whether PHEX binds specifically to MEPE, (2) whether the binding involves the ASARM motif region, and (3) whether free ASARM peptide affects mineralization in vivo in mice. Protein interactions between MEPE and recombinant soluble PHEX (secPHEX) were measured using surface plasmon resonance (SPR). Briefly, secPHEX, MEPE, and control protein (IgG) were immobilized on a Biacore CM5 sensor chip, and SPR experiments were performed on a Biacore 3000 high-performance research system. Pure secPHEX was then injected at different concentrations, and interactions with immobilized proteins were measured. To determine MEPE sequences interacting with secPHEX, the inhibitory effects of MEPE-ASARM peptides (phosphorylated and nonphosphorylated), control peptides, and MEPE midregion RGD peptides on secPHEX binding to chip-immobilized MEPE were measured. ASARM peptide and etidronate-mediated mineralization inhibition in vivo and in vitro were determined by quenched calcein fluorescence in hind limbs and calvariae in mice and by histological Sanderson stain. A specific, dose-dependent and Zn-dependent protein interaction between secPHEX and immobilized MEPE occurs (EC50 of 553 nM). Synthetic MEPE PO4-ASARM peptide inhibits the PHEX-MEPE interaction (K(D(app)) = 15 uM and B(max/inhib) = 68%). In contrast, control and MEPE-RGD peptides had no effect. Subcutaneous administration of ASARM peptide resulted in marked quenching of fluorescence in calvariae and hind limbs relative to vehicle controls indicating impaired mineralization. Similar results were obtained with etidronate. Sanderson-stained calvariae also indicated a marked increase in unmineralized osteoid with ASARM peptide and etidronate groups. We conclude that PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif, and the ASARM peptide inhibits mineralization in vivo. The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in HYP.