Clarence D. Lin

Decreased Circulating Progenitor Cell Number and Failed Mechanisms of Stromal Cell-Derived Factor-1α Mediated Bone Marrow Mobilization Impair Diabetic Tissue Repair

OBJECTIVE Progenitor cells (PCs) contribute to postnatal neovascularization and tissue repair. Here, we explore the mechanism contributing to decreased diabetic circulating PC number and propose a novel treatment to restore circulating PC number, peripheral neovascularization, and tissue healing. RESEARCH DESIGN AND METHODS Cutaneous wounds were created on wild-type (C57BL/J6) and diabetic (Leprdb/db) mice. Blood and bone marrow PCs were collected at multiple time points. RESULTS Significantly delayed wound closure in diabetic animals was associated with diminished circulating PC number (1.9-fold increase vs. 7.6-fold increase in lin−/sca-1+/ckit+ in wild-type mice; P < 0.01), despite adequate numbers of PCs in the bone marrow at baseline (14.4 ± 3.2% lin−/ckit+/sca1+ vs. 13.5 ± 2.8% in wild-type). Normal bone marrow PC mobilization in response to peripheral wounding occurred after a necessary switch in bone marrow stromal cell-derived factor-1α (SDF-1α) expression (40% reduction, P < 0.01). In contrast, a failed switch mechanism in diabetic bone marrow SDF-1α expression (2.8% reduction) resulted in impaired PC mobilization. Restoring the bone marrow SDF-1α switch (54% reduction, P < 0.01) with plerixafor (Mozobil, formerly known as AMD3100) increased circulating diabetic PC numbers (6.8 ± 2.0-fold increase in lin−/ckit+, P < 0.05) and significantly improved diabetic wound closure compared with sham-treated controls (32.9 ± 5.0% vs. 11.9 ± 3% at day 7, P > 0.05; 73.0 ± 6.4% vs. 36.5 ± 7% at day 14, P < 0.05; and 88.0 ± 5.7% vs. 66.7 ± 5% at day 21, P > 0.05, respectively). CONCLUSIONS Successful ischemia-induced bone marrow PC mobilization is mediated by a switch in bone marrow SDF-1α levels. In diabetes, this switch fails to occur. Plerixafor represents a potential therapeutic agent for improving ischemia-mediated pathology associated with diabetes by reducing bone marrow SDF-1α, restoring normal PC mobilization and tissue healing.

Decreased Circulating Progenitor Cell Number and Failed Mechanisms of SDF-1α Mediated Bone Marrow Mobilization Impair Diabetic Tissue Repair

OBJECTIVE Progenitor cells (PCs) contribute to postnatal neovascularization and tissue repair. Here, we explore the mechanism contributing to decreased diabetic circulating PC number and propose a novel treatment to restore circulating PC number, peripheral neovascularization, and tissue healing. RESEARCH DESIGN AND METHODS Cutaneous wounds were created on wild-type (C57BL/J6) and diabetic (Leprdb/db) mice. Blood and bone marrow PCs were collected at multiple time points. RESULTS Significantly delayed wound closure in diabetic animals was associated with diminished circulating PC number (1.9-fold increase vs. 7.6-fold increase in lin−/sca-1+/ckit+ in wild-type mice; P < 0.01), despite adequate numbers of PCs in the bone marrow at baseline (14.4 ± 3.2% lin−/ckit+/sca1+ vs. 13.5 ± 2.8% in wild-type). Normal bone marrow PC mobilization in response to peripheral wounding occurred after a necessary switch in bone marrow stromal cell-derived factor-1α (SDF-1α) expression (40% reduction, P < 0.01). In contrast, a failed switch mechanism in diabetic bone marrow SDF-1α expression (2.8% reduction) resulted in impaired PC mobilization. Restoring the bone marrow SDF-1α switch (54% reduction, P < 0.01) with plerixafor (Mozobil, formerly known as AMD3100) increased circulating diabetic PC numbers (6.8 ± 2.0-fold increase in lin−/ckit+, P < 0.05) and significantly improved diabetic wound closure compared with sham-treated controls (32.9 ± 5.0% vs. 11.9 ± 3% at day 7, P > 0.05; 73.0 ± 6.4% vs. 36.5 ± 7% at day 14, P < 0.05; and 88.0 ± 5.7% vs. 66.7 ± 5% at day 21, P > 0.05, respectively). CONCLUSIONS Successful ischemia-induced bone marrow PC mobilization is mediated by a switch in bone marrow SDF-1α levels. In diabetes, this switch fails to occur. Plerixafor represents a potential therapeutic agent for improving ischemia-mediated pathology associated with diabetes by reducing bone marrow SDF-1α, restoring normal PC mobilization and tissue healing.

Topical Lineage-negative Progenitor-cell Therapy for Diabetic Wounds

Background: Impaired diabetic wound healing is due, in part, to defects in mesenchymal progenitor cell tracking. Theoretically, these defects may be overcome by administering purified progenitor cells directly to the diabetic wound. The authors hypothesize that these progenitor cells will differentiate into endothelial cells, increase wound vascularity, and improve wound healing. Methods: Lineage-negative progenitor cells were isolated from wild-type murine bone marrow by magnetic cell sorting, suspended in a collagen matrix, and applied topically to full-thickness excisional dorsal cutaneous wounds in diabetic mice. Application of lineage-positive hematopoietic cells or acellular collagen matrix served as comparative controls (n = 16 for each group; n = 48 total). Time to closure and percentage closure were calculated by morphometry. Wounds were harvested at 7, 14, 21, and 28 days and then processed, sectioned, stained (lectin/DiI and CD31), and vascularity was quantified. Results: Wounds treated with lineage-negative cells demonstrated a significantly decreased time to closure (14 days) compared with lineage-positive (21 days, p = 0.013) and collagen controls (28 days, p = 0.004), and a significant improvement in percentage closure at 14 days compared with the lineage-positive group (p < 0.01) and the collagen control (p < 0.01). Fluorescently tagged lineage-negative cells remained viable in the wound for 28 days, whereas lineage-positive cells were not present after 7 days. Lineage-negative, but not lineage-positive, cells differentiated into endothelial cells. Vascular density and vessel cross-sectional area were significantly higher in lineage-negative wounds. Conclusion: Topical progenitor-cell therapy successfully accelerates diabetic wound closure and improves wound vascularity.

Establishment of a Critical-Sized Alveolar Defect in the Rat : A Model for Human Gingivoperiosteoplasty

Background: Despite technical advancement, treatment of congenital alveolar clefts has remained controversial. Currently, primary alveolar cleft repair (i.e., gingivoperiosteoplasty) has a 41 to 73 percent success rate. However, the remaining patients have persistent alveolar bone defects requiring secondary grafting procedures. Morbidity of secondary procedures includes pain, graft resorption, extrusion or infection, and graft or tooth loss. The authors present a novel rat alveolar defect model designed to facilitate investigation of therapeutics aimed at improving bone formation following primary alveolar cleft repair in humans. Methods: Sixteen 8-week-old Sprague-Dawley rats underwent creation of a 7 × 4 × 3-mm complete alveolar defect from the maxillary incisors to the zygomatic arch. Four animals were humanely killed at each of the following time points: 0, 4, 8, and 12 weeks. Morphometric analysis of the alveolar defect was determined by means of micro-computed tomography and histology. Results: Micro-computed tomography demonstrated that new bone filled 43 ± 5.6 percent of the alveolar defect at 4 weeks, 53 ± 8.3 percent at 8 weeks, and 48 ± 3.5 percent at 12 weeks. Histologically, at 4 weeks, proliferating fibroblasts and polymorphonuclear cells were scattered throughout the disorganized collagen in the intercalary gap. By 8 weeks, nascent woven bone spicules extended from the edges of the defect. At 12 weeks, the woven spicules had remodeled, with scant additional bone deposition. Conclusion: This model creates a critical-size alveolar defect that is similar in size and location to human alveolar defects and is suitable for studying proposed therapeutics.

Scaffold-based rhBMP-2 therapy in a rat alveolar defect model: implications for human gingivoperiosteoplasty.

Background: Primary alveolar cleft repair has a 41 to 73 percent success rate. Patients with persistent alveolar defects require secondary bone grafting. The authors investigated scaffold-based therapies designed to augment the success of alveolar repair. Methods: Critical-size, 7 × 4 × 3-mm alveolar defects were created surgically in 60 Sprague-Dawley rats. Four scaffold treatment arms were tested: absorbable collagen sponge, absorbable collagen sponge plus recombinant human bone morphogenetic protein-2 (rhBMP-2), hydroxyapatite–tricalcium phosphate, hydroxyapatite–tricalcium phosphate plus rhBMP-2, and no scaffold. New bone formation was assessed radiomorphometrically and histomorphometrically at 4, 8, and 12 weeks. Results: Radiomorphometrically, untreated animals formed 43 ± 6 percent, 53 ± 8 percent, and 48 ± 3 percent new bone at 4, 8, and 12 weeks, respectively. Animals treated with absorbable collagen sponge formed 50 ± 6 percent, 79 ± 9 percent, and 69 ± 7 percent new bone, respectively. Absorbable collagen sponge plus rhBMP-2–treated animals formed 49 ± 2 percent, 71 ± 6 percent, and 66 ± 7 percent new bone, respectively. Hydroxyapatite–tricalcium phosphate treatment stimulated 69 ± 12 percent, 86 ± 3 percent (p < 0.05), and 87 ± 14 percent new bone, respectively. Histomorphometry demonstrated an increase in bone formation in animals treated with hydroxyapatite–tricalcium phosphate plus rhBMP-2 (p < 0.05; 4 weeks) compared with empty scaffold. Conclusions: Radiomorphometrically, absorbable collagen sponge and hydroxyapatite–tricalcium phosphate scaffolds induced more bone formation than untreated controls. The rhBMP-2 added a small but significant histomorphometric osteogenic advantage to the hydroxyapatite–tricalcium phosphate scaffold.

Combination Therapy Accelerates Diabetic Wound Closure

Background Non-healing foot ulcers are the most common cause of non-traumatic amputation and hospitalization amongst diabetics in the developed world. Impaired wound neovascularization perpetuates a cycle of dysfunctional tissue repair and regeneration. Evidence implicates defective mobilization of marrow-derived progenitor cells (PCs) as a fundamental cause of impaired diabetic neovascularization. Currently, there are no FDA-approved therapies to address this defect. Here we report an endogenous PC strategy to improve diabetic wound neovascularization and closure through a combination therapy of AMD3100, which mobilizes marrow-derived PCs by competitively binding to the cell surface CXCR4 receptor, and PDGF-BB, which is a protein known to enhance cell growth, progenitor cell migration and angiogenesis. Methods and Results Wounded mice were assigned to 1 of 5 experimental arms (n = 8/arm): saline treated wild-type, saline treated diabetic, AMD3100 treated diabetic, PDGF-BB treated diabetic, and AMD3100/PDGF-BB treated diabetic. Circulating PC number and wound vascularity were analyzed for each group (n = 8/group). Cellular function was assessed in the presence of AMD3100. Using a validated preclinical model of type II diabetic wound healing, we show that AMD3100 therapy (10 mg/kg; i.p. daily) alone can rescue diabetes-specific defects in PC mobilization, but cannot restore normal wound neovascularization. Through further investigation, we demonstrate an acquired trafficking-defect within AMD3100-treated diabetic PCs that can be rescued by PDGF-BB (2 μg; topical) supplementation within the wound environment. Finally, we determine that combination therapy restores diabetic wound neovascularization and accelerates time to wound closure by 40%. Conclusions Combination AMD3100 and PDGF-BB therapy synergistically improves BM PC mobilization and trafficking, resulting in significantly improved diabetic wound closure and neovascularization. The success of this endogenous, cell-based strategy to improve diabetic wound healing using FDA-approved therapies is inherently translatable.

Circulating Endothelial Progenitor Cells and Diabetic Wound Healing

METHODS: Full-thickness wounds were made on the dorsum of wild-type (wt) and type II Leprdb/db mice. Mice were randomized into 5 experimental arms (n=8/arm): untreated wt (WT), untreated db (DB), AMD3100-treated db (A+), PDGFBB-treated db (P+), and AMD3100/PDGF-BB-treated db (A+P+). Treated mice received daily AMD3100 (10mg/kg; i.p.) and/or PDGF-BB (2μg; topical) beginning on post-wounding day 3 and continuing until wound closure. Wound closure was assessed photometrically. Circulating (c)EPC number was determined by FACS. Wound vascularity (vessels/hpf) was assessed by CD31 immunofluorescence. Wound fibroblast and EPC function were assessed in vitro in the presence of AMD3100 (5-50ng/mL).