Alexander C. Allori

Biological basis of bone formation, remodeling, and repair-part II: extracellular matrix.

The bony biochemical environment is a complex system that permits and promotes cellular functions that lead to matrix production and ossification. In Part I of this review, we discussed the important actions of signaling molecules, including hormones, cytokines, and growth factors. Here, we review other constituents of the extracellular matrix, including minerals, fibrinous and nonfibrinous proteins, and enzymes such as the matrix metalloproteinases. We conclude with a discussion of the role of biochemical modulation in endogenous and exogenous tissue engineering.

Biological Basis of Bone Formation, Remodeling, and Repair—Part I: Biochemical Signaling Molecules

The bony biochemical environment is an active and dynamic system that permits and promotes cellular functions that lead to matrix production and ossification. Each component is capable of conveying important regulatory cues to nearby cells, thus effecting gene expression and changes at the cytostructural level. Here, we review the various signaling molecules that contribute to the active and dynamic nature of the biochemical system. These components include hormones, cytokines, and growth factors. We describe their role in regulating bone metabolism. Certain growth factors (i.e., TGF-beta, IGF-1, and VEGF) are described in greater detail because of their potential importance in developing successful tissue-engineering strategies.

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.

Biological Basis of Bone Formation, Remodeling, and Repair—Part III: Biomechanical Forces

While it has been long appreciated that biomechanical forces are involved in bone remodeling and repair, the actual mechanism by which a physical force is translated to the corresponding intracellular signal has largely remained a mystery. To date, most biomechanical research has concentrated upon the effect on bone morphology and architecture, and it is only recently that the complex cellular and molecular pathways involved in this process (called mechanotransduction) are being described. In this paper, we review the current understanding of bone mechanobiology and highlight the implications for clinical medicine and tissue engineering research.

A Novel Flow-Perfusion Bioreactor Supports 3D Dynamic Cell Culture

Background. Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm). A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm) scaffolds. Methods. Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. Results. By day 8, static scaffolds had a periphery cell density of 67% ± 5.0%, while in the core it was 0.3% ± 0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94% ± 8.3% and core density of 76% ± 3.1% at day 8. Conclusions. Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

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.

Progenitor cell mobilization enhances bone healing by means of improved neovascularization and osteogenesis.

Background: Although bone repair is a relatively efficient process, a significant portion of patients fail to heal their fractures. Because adequate blood supply is essential to osteogenesis, the authors hypothesize that augmenting neovascularization by increasing the number of circulating progenitor cells will improve bony healing. Methods: Bilateral full-thickness defects were created in the parietal bones of C57 wild-type mice. Intraperitoneal AMD3100 (n = 33) or sterile saline (n = 33) was administered daily beginning on postoperative day 3 and continuing through day 18. Circulating progenitor cell number was quantified by fluorescence-activated cell sorting. Bone regeneration was assessed with micro–computed tomography. Immunofluorescent CD31 and osteocalcin staining was performed to assess for vascularity and osteoblast density. Results: AMD3100 treatment increased circulating progenitor cell levels and significantly improved bone regeneration. Calvarial defects of AMD3100-treated mice demonstrated increased vascularity and osteoblast density. Conclusions: Improved bone regeneration in this model was associated with elevated circulating progenitor cell number and subsequently improved neovascularization and osteogenesis. These findings highlight the importance of circulating progenitor cells in bone healing and may provide a novel therapy for bone regeneration.

Periosteum-guided prefabrication of vascularized bone of clinical shape and volume.

Background: Large craniofacial skeletal defects require complex reconstruction. Vascularized tissue transfer is the current standard in treatment, but these operations are technically difficult and associated with donor-site morbidity. Guided flap prefabrication offers a technique for endogenously engineering vascularized composite tissues with complex three-dimensional structure. This study evaluates the relationship between implantation time and tissue structure for generating tissues of clinically relevant volume and structure. Methods: Twenty skeletally mature domestic sheep were implanted with poly(methyl methacrylate) chambers designed to mimic the size and shape of the mental protuberance of the mandible. Each chamber was filled with morcellized bone graft and implanted with the open face apposed to the cambium layer of the rib periosteum. Chambers were harvested at 3, 6, 9, 12, and 24 weeks, and the tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphometric parameters. Results: Histologically, active endochondral, direct, and appositional bone formation was observed. Calcified tissue area and new bone formation increased for each time point up to 12 weeks of implantation. The tissues formed maintained volumetric and geometrical structure consistent with the chamber up to 9 weeks after implantation. Significant decreases in total volume and agreement with chamber geometry were observed at 12 and 24 weeks. Conclusions: Periosteum-guided tissue prefabrication was found to be an effective means of engineering three-dimensional vascularized bone of clinical size and shape. The optimal duration of incubation before significant volume loss occurs is 9 weeks in this large-animal model.

Comparison of guided bone formation from periosteum and muscle fascia.

Background: Muscle fascia and periosteum have been used clinically to guide prefabrication of vascularized bone flaps for reconstruction of complex three-dimensional tissues. Although it seems that both locations have the capacity to generate vascularized bone, there have been no studies that directly compare different implantation sites. The authors performed a rigorous, quantitative, histomorphometric comparison of bone prefabrication in a large-animal model comparing graft implanted against muscle fascia and periosteum. Methods: Twenty skeletally mature domestic sheep were implanted with rectangular chambers containing equal weights of morcellized bone graft. Two chambers were implanted into each sheep, one with the open face apposed to the cambium layer of the rib periosteum and the other with the open face apposed to the fascia of the latissimus dorsi muscle. Animals were euthanized at 3, 6, 9, 12, and 24 weeks and the chambers were harvested. Tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphology. Results: There were no differences in volume or shape of tissue formed in the chambers. However, chambers in contact with fascia consisted almost entirely of fibrovascular tissue, with progressive resorption of the morcellized bone graft and little evidence of new bone. Chambers in contact with periosteum showed active endochondral, direct, and appositional bone formation over time, with increasing calcified tissue area and new bone formation. Conclusions: Both periosteum and muscle fascia were able to vascularize bone grafts, but bone formation was higher in the periosteum. The periosteum appears to be a more suitable foundation from which to promote flap prefabrication.

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.