Assessment of Hedgehog Signaling Pathway Activation for Craniofacial Bone Regeneration in a Critical-Sized Rat Mandibular Defect.

IMPORTANCE: Osseous craniofacial defects are currently reconstructed with bone grafting, rigid fixation, free tissue transfer, and/or recombinant human bone morphogenetic protein 2. Although these treatment options often have good outcomes, they are associated with substantial morbidity, and many patients are not candidates for free tissue transfer. OBJECTIVE: To assess whether polysaccharide-based scaffold (PS) constructs that are cross-linked with smoothened agonist (SAG), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 6 (BMP-6) would substantially increase bone regeneration. DESIGN, SETTING, AND PARTICIPANTS: This animal model study was conducted at the University of Virginia School of Medicine Cui Laboratory from March 1, 2017, to June 30, 2017. Thirty-three 10-week-old female Lewis rats were acquired for the study. Bilateral nonsegmental critical-sized defects were created in the angle of rat mandibles. The defects were either left untreated or filled with 1 of the 9 PSs. The rats were killed after 8 weeks, and bone regeneration was evaluated using microcomputed tomographic imaging and mechanical testing. Analysis of variance testing was used to compare the treatment groups. MAIN OUTCOMES AND MEASURES: Blinded analysis and computer analysis of the microcomputed tomographic images were used to assess bone regeneration. RESULTS: In the 33 female Lewis rats, minimal healing was observed in the untreated mandibles. Addition of SAG was associated with increases in bone regeneration and bone density in all treatment groups, and maximum bone healing was seen in the group with BMP-6, VEGF, and SAG cross-linked to PS. For each of the 5 no scaffold group vs BMP-6, VEGF, and SAG cross-linked to PS group comparisons, mean defect bone regeneration was 4.14% (95% CI, 0.94%-7.33%) vs 66.19% (95% CI, 54.47%-77.90%); mean bone volume, 14.52 mm3 (95% CI, 13.07-15.97 mm3) vs 20.87 mm3 (95% CI, 14.73- 27.01 mm3); mean bone surface, 68.97 mm2 (95% CI, 60.08-77.85 mm2) vs 96.77 mm2 (95% CI, 76.11-117.43 mm2); mean ratio of bone volume to total volume, 0.11 (95% CI, 0.10-0.11) vs 0.15 (95% CI, 0.10-0.19); and mean connectivity density 0.03 (95% CI, 0.02-0.05) vs 0.32 (95% CI, 0.25-0.38). On mechanical testing, mandibles with untreated defects broke with less force than control mandibles in which no defect was made, although this force did not reach statistical significance. No significant difference in force to fracture was observed among the treatment groups. CONCLUSIONS AND RELEVANCE: In this rat model study, activation of the hedgehog signaling pathway using smoothened agonist was associated with increased craniofacial bone regeneration compared with growth factors alone, including US Food and Drug Administration-approved recombinant human bone morphogenetic protein 2. Pharmaceuticals that target this pathway may offer a new reconstructive option for bony craniofacial defects as well as nonunion and delayed healing fractures. LEVEL OF EVIDENCE: NA.

Peptides from phage display library modulate gene expression in mesenchymal cells and potentiate osteogenesis in unicortical bone defects.

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

Growth and differentiation factor-5 (GDF-5) stimulates osteogenic differentiation and increases vascular endothelial growth factor (VEGF) levels in fat-derived stromal cells in vitro.

Fat-derived adult mesenchymal stem cells can differentiate into different phenotypes reflecting their potential to regenerate various skeletal tissues. These properties together with the association of adipose with skeletal tissues formed the basis of our study to establish an experimental model for using fat-derived stromal cells to undergo osteogenic differentiation in vitro under the influence of either growth and differentiation factor-5 (GDF-5) or bone morphogenetic protein-2 (BMP-2). Members of the BMP/GDF family of proteins are known for their ability to elicit skeletal morphogenesis, but little is known about the mechanism whereby these morphogens exert their effect on the osteogenic differentiation of fat-derived stromal cells. We compared the effects of GDF-5 and BMP-2 in their recombinant forms to qualitatively and quantitatively determine their influence on the osteogenic differentiation of fat derived stromal cells by examining the effects on mineralization, extracellular matrix, cell proliferation, biochemistry, and gene expression. We identified that GDF-5 not only promotes osteogenic differentiation of rat fat-derived stromal cells, but also may promote angiogenic activity of stromal cells by increasing vascular endothelial growth factor (VEGF) gene expression in vitro. These data suggest that several distinct regulatory mechanisms may exist in association with osteogenic differentiation.

Recombinant growth/differentiation factor-5 stimulates osteogenic differentiation of fat-derived stromal cells in vitro.

Fat-derived stromal cells can differentiate into various skeletal tissues. Currently the mechanism that determines whether stromal cells differentiate into osteoblasts is unclear and the role of growth/differentiation factor (GDF)-5 in differentiation of fat-derived stromal cells is not fully understood. It appears that the differentiation of stromal cells is greatly enhanced by GDF-5 that plays a role in a variety of musculoskeletal processes such as joint formation, tendon maintenance, and bone formation. Our study showed that GDF-5 promotes the differentiation of rat fat-derived stromal cells into osteogenic lineages in vitro. Furthermore, these findings were confirmed by histology, biochemical assay for alkaline phosphatase activity, and analysis of gene expression. The ability to preferentially stimulate fat-derived stromal cells down the osteogenic pathway holds significance in a variety of clinical scenarios.

An adenosine A2A receptor agonist reduces interleukin-8 expression and glycosaminoglycan loss following septic arthrosis.

The purpose of this study was to determine whether an adenosine A(2A) receptor agonist (ATL146e) might augment the current treatment regimen of antibiotics plus irrigation and debridement to prevent the arthritic effects associated with joint sepsis. Staphylococcus aureus bacteria were injected into knees of rabbits, which were divided into 4 treatment groups (12 rabbits per group): no treatment, ATL146e only, antibiotics only, or antibiotics plus ATL146e. Analysis at days 1, 3, and 7 consisted of gross joint appearance, synovial fluid, serum, histologic, immunohistochemical, and biochemical analysis. Synovial fluid cultures at day 7 were negative in all antibiotic and antibiotic plus ATL146e treated knees indicating clearance of bacteria. Average WBC counts from synovial fluid aspirates significantly decreased with treatment of antibiotics alone and antibiotics plus ATL146e. Treatment with antibiotics plus ATL146e significantly decreased the Interleukin-8 content when compared to other treatment groups (p<0.001) indicating inflammatory response suppression. Histologic grading resulted in notably improved scores in the antibiotics plus ATL146e group compared to other treatment groups (p < or =0.001). Glycosaminoglycan assay values were significantly greater in the ATL146e plus antibiotics group compared to the untreated control group (p<0.04) indicating chondroprotection. The results of this study indicate that administration of an adenosine A(2A) agonist in combination with antibiotic therapy diminishes joint WBC chemotaxis and reduces joint inflammation, while not compromising the clearance of intraarticular bacteria in a rabbit model. Early bacterial clearance with modulation of the inflammatory response appears to prevent the early degradative effects of joint sepsis.

A new method of selecting Schwann cells from adult mouse sciatic nerve.

We describe a method of using laminin for the selection and purification of Schwann cells in vitro. We also studied the viability of the selected cells suspended in alginate beads both in vitro and in vivo. We observed that the homogeneity of the Schwann cell culture increased with each round of laminin selection and reached 85-90% after five passages. The viability of cells after incubation within an alginate bead in vivo was between 73 and 76% compared with greater than 90% viability for cells that were maintained in monolayer culture. This new method of serial selection using laminin-coated surfaces has optimized the purification of a Schwann cell culture expanded from cells harvested from the adult sciatic nerve of a mouse. This method has the advantage of being technically easier than other methods described and results in a Schwann cell culture that is 80-90% homogenous.

Collagen and proteoglycan abnormalities in the GDF-5-deficient mice and molecular changes when treating disk cells with recombinant growth factor.

STUDY DESIGN: A magnetic resonance image, histologic, biochemical, and gene expression study was conducted to characterize the effects of growth and development factor-5 (GDF-5) deficiency on the health of the intervertebral disc. OBJECTIVE: To determine the effect of GDF-5 deficiency on extracellular matrix and gene expression on the intervertebral disc. SUMMARY OF BACKGROUND DATA: Developmental and degenerative changes in intervertebral disc are not fully understood. Molecular abnormalities and spontaneous mutations that lead to the deficiency in a normal protein have been useful in understanding the function of certain molecules and the role they play in the structure and health of certain tissues. Although the role of GDF-5 in the disc has not been elucidated, this factor may have an important role in the disc as a result of the well-documented effect of GDF-5 in other chondrogenic tissues. METHODS.: Intervertebral discs of 20-week-old GDF-5 (-/-) and (+/+) mice were examined radiographically, histologically, biochemically, and with gene expression studies. Cells isolated from GDF-5-deficient mouse discs were treated with recombinant GDF-5 and gene expression was subsequently analyzed. RESULTS: GDF-5 (-/-) mice demonstrated significantly lower T2-weighted signal intensity in the central region of their lumbar discs, and disc histology revealed loss of the normal lamellar architecture of the anulus fibrosus and a shrunken, disorganized nucleus pulposus. Biochemical analysis revealed decreased proteoglycan content but no appreciable change in total collagen content of the discs. Significant downregulation of both aggrecan and type II collagen mRNA, without an appreciable change in type I collagen expression, was noted on gene expression studies. Recombinant GDF-5 treatment of disc cells from the GDF-5-deficient mice resulted in a dose-dependent upregulation of the aggrecan and type II collagen genes. CONCLUSION: The intervertebral disc is markedly affected by GDF-5 deficiency. This relatively simple (single gene) system with a known molecular defect may be useful in studies designed to define the response of the intervertebral disc to treatment with growth factor in vivo.

A fibronectin fragment stimulates intervertebral disc degeneration in vivo.

STUDY DESIGN: A radiographic, histologic, biochemical, and gene expression study was conducted in vivo in a rabbit model to determine the effect of injection of the N-terminal 30 kDa fibronectin fragment (Fn-f) into the intervertebral disc along with various control substances. OBJECTIVE: To determine if the Fn-f is able to induce disc degeneration in vivo. SUMMARY OF BACKGROUND DATA: Animal models of disc degeneration are crucial to defining the molecular events involved in disc degeneration. Although spontaneous and induced models of disc degeneration have been described, none is ideal for molecular studies. A better understanding of disc degeneration at the molecular level is necessary to promote rational design of therapies for degenerative disc disease. MATERIALS AND METHODS: Thirty-one New Zealand white rabbits underwent injection of Fn-f and control substances into the central region of separate lumbar discs using a fine needle. Euthanasia was performed at the 2-, 4-, 8-, 12-, and 16-week time points and the discs were examined radiographically, histologically, biochemically, and with gene expression. RESULTS: Radiographs demonstrated anterior osteophyte formation at Fn-f-injected disc spaces by the 12-week time point. Histology demonstrated a progressive loss of the normal architecture of the nucleus pulposus and anulus fibrosus over the 16-week study period. A progressive loss of proteoglycans was documented using GAG assay but total collagen did not appear to change appreciably. Gene expression studies demonstrated a significant down-regulation of both aggrecan and type II collagen mRNA between the 8- and 16-week time points. CONCLUSION: Fn-f appears to induce a progressive degenerative process within the intervertebral disc after injection that resembles degenerative disc disease. This model has several significant advantages for the study of disc degeneration at the molecular level. Further studies are warranted to elucidate the mechanism and molecular events associated with Fn-f-mediated disc degeneration.