Sean A.F. Peel

Closure of rabbit calvarial critical-sized defects using protective composite allogeneic and alloplastic bone substitutes.

This study evaluated the repair of critical-sized cranial vault defects in thirty New Zealand white rabbits using various allogeneic and alloplastic bone substitutes designed to provide mechanical protection to the brain as well as osteoinductivity. The strategies employed included demineralized bone matrix (DBM), a putty used in combination with a rigid resorbable plating system as a protective covering and calcium phosphate cement (CPC) combined with native partially purified bone morphogenetic protein (BMP). Bilateral critical-sized defects measuring 15 mm in diameter were created in the parietal bones of 30 adult male New Zealand white rabbits. They were divided into three groups with ten animals in each. Group 1 had one defect left unfilled as a control while autogenous bone was placed in the defect on the other side. In Group 2 a rigid resorbable copolymer membrane, Lactosorb (Lorenz Surgical, Jacksonville, Florida), was placed over both defects to cover them and protect the underlying tissues. The pericranial aspect of one defect was left unfilled while the other defect was filled with DBM putty. Group 3 had a CPC, Mimix (Lorenz Surgical, Jacksonville, Florida), placed into one of the defects while the defect on the other side was filled with the same CPC in combination with BMP in a concentration of 25 mg/mL. Bone healing was assessed clinically, radiographically, and histomorphometrically. All unfilled controlled defects, the defects covered with the resorbable Lactosorb membrane and those filled with calcium phosphate cement alone, healed with a fibrous scar. Defects reconstructed with DBM putty in combination with the resorbable Lactosorb membrane and calcium phosphate in combination with BMP healed with bone bridging the entire defect. This was obvious radiographically where the defects appeared completely filled with a dense radiopaque tissue. Histological analysis demonstrated that specimens where DBM putty was used in combination with the resorbable Lactosorb membrane had 67.7% new bone fill at 6 weeks and 84.0% at 12 weeks. Resorption of DBM particles was evidenced by the presence of osteoclastic activity and by the significant decrease in the size of the demineralized bone particles. In the calcium phosphate groups where BMP was added to the bioimplant there was 45.8% new bone formation at 12 weeks. The utilization of a composite consisting of DBM with resorbable Lactosorb membrane or a composite of calcium phosphate cement composite with BMP promoted complete closure of critical-sized calvarial defects in New Zealand white rabbits with viable new bone at 12 weeks. The complete bone bridging observed with these composites suggests that they could be used to enhance the protection of intracranial contents following craniofacial surgical procedures.

In search of the ideal bone morphogenetic protein delivery system: in vitro studies on demineralized bone matrix, purified, and recombinant bone morphogenetic protein.

The clinical use of recombinant bone morphogenetic protein (rBMP) is limited by the lack of a suitable delivery system. The bone morphogenetic protein (BMP) delivery system provided by nature is highly effective, and by studying purified BMP (BMP/NCP) and demineralized bone matrix (DBM), it may be possible to learn how to emulate natures success. The current study used an in vitro muscle cell model to study the activity of BMP/NCP and DBM and the effects of extracellular matrix on BMP activity. C2C12 cells transiently exposed to recombinant human BMP-4 (rhBMP-4) rapidly increased their alkaline phosphatase (AP) activity to day 5, after which it steadily declined. Cells exposed to BMP/NCP or DBM continued to increase their AP activity over the 14-day culture. If BMP/NCP was treated to remove a 22-kd protein, it became water-soluble and exhibited a similar activity pattern to rhBMP-4. Cells cultured on collagen type I, fibronectin, and hyaluronic-coated surfaces demonstrated increased AP activity when exposed to rhBMP-4 or BMP/NCP compared with cells cultured on bovine serum albumin or poly-l-lysine. These results suggest that the natural BMP delivery system operates both by binding to the BMP molecule and slowly releasing it into the extracellular milieu and by interacting with the responding cells through cell-matrix receptors to enhance the cellular response to BMP.

Effect of hyperbaric oxygen on demineralized bone matrix and biphasic calcium phosphate bone substitutes.

OBJECTIVES The aim of this study was to assess the possible effect of hyperbaric oxygen (HBO) on the healing of critical-sized defects that were grafted with demineralized bone matrix (DBM) combined with Pluronic F127 (F127) to form a gel or putty, or a commercially available biphasic calcium phosphate (BCP), mixed either with blood or F127 to form a putty. STUDY DESIGN Twenty New Zealand White rabbits were randomly divided into 2 groups of 10 animals each. Bilateral 15-mm calvarial defects were created in the parietal bones of each animal, resulting in 40 critical-sized defects. Group I defects were grafted with either DBM putty or DBM gel. Group II defects were grafted with either BCP or BCP putty. Five animals from each group received HBO treatment (100% oxygen, at 2.4 ATA) for 90 minutes per day 5 days a week for 4 weeks. The other 5 animals in each group served as a normobaric (NBO) controls, breathing only room air. All animals were humanely killed at 6 weeks. The calvariae were removed and analyzed by micro computed tomography (mCT) and histomorphometry. RESULTS mCT analysis indicated a higher bone mineral content (BMC, P < .05), bone volume fraction (BVF; P < .001), and bone mineral density (BMD; P < .001) of the defects grafted with BCP rather than DBM. Furthermore, the voxels that were counted as bone had a higher tissue mineral density (TMD) in the BCP- than in the DBM-filled defects (P < .001). Histologically complete bony union over the defects was observed in all specimens. Histomorphometric analysis showed that DBM-filled defects had more new bone (P < .007) and marrow (P < .001), and reduced fibrous tissue compared with the BCP defects (P < .001) under NBO conditions. HBO treatment reduced the amount of fibrous tissue in BCP filled defects (P < .05), approaching levels similar to that in matching DBM-filled defects. HBO also resulted in a small but significant increase in new bone in DBM-grafted defects (P < .05). CONCLUSION Use of DBM or BCP promoted healing in these critical-sized defects. Hyperbaric oxygen therapy resulted in a slight increase in new bone in DBM-grafted defects and much larger reduction in fibrous tissue and matching increases in marrow in BCP-grafted defects, possibly through increased promotion of angiogenesis.

Role of bovine bone morphogenetic proteins in bone matrix protein and osteoblast-related gene expression during rat bone marrow stromal cell differentiation.

Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway evaluating the ability of certain BMPs to promote bone graft and fracture healing. To observe the mechanism of osteoinductive and bone formation, 100 ug of bovine BMP was tested during osteogenic differentiation of rat bone marrow stromal cells (MSCs) and C2C12 line culture for 14 and 28 days. We examined alkaline phosphatase (ALP) by assay, immunohistochemical studies for bone matrix proteins, and mRNA expression of bone matrix proteins and osteoblast-related analysis by reverse-transcription polymerase chain reaction. ALP activity in MSC cultures was elevated by bovine BMP by two to fivefold (P < 0.05-0.001). DNA and protein content increased over 14 days. BMP significantly increased the mRNA expression of type I collagen, ALP, osterix, osteocalcin, osteopontin, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-A, and parathyroid hormone receptor time dependently during the osteoblastic differentiation. There was no markedly enhanced mRNA expression of bone sialoprotein (BSP) and glyceraldehyde-3-phosphate dehydrogenase compared with that of control. Immunohistochemical results also showed BMP increased immunoreactive positivity of type I collagen, osteocalcin, osteonectin, osteopontin, and BSP during the C2C12 differentiation. These data indicated that BMP enhances our ability to stimulate the differentiation of osteoblast-like cells and increases osteoinductivity, bone matrix protein formation and mineralization, angiogenesis, and chondrogenesis during osteoblast progenitor cell differentiation in vitro and that the role of chondrogenic is weak.

Hyperbaric oxygen results in increased vascular endothelial growth factor (VEGF) protein expression in rabbit calvarial critical-sized defects

BACKGROUND Hyperbaric oxygen therapy (HBO) promotes osseous healing, however the mechanism by which this occurs has not been elucidated. HBO may promote angiogenesis, which is vital for bone healing. Vascular endothelial growth factor (VEGF) is one of the key factors that stimulates angiogenesis. OBJECTIVE The objective of this study was to investigate whether HBO altered VEGF expression during bone healing. METHODS AND MATERIALS Archived samples from calvarial defects of rabbits exposed to HBO (2.4 ATA, 90 minutes a day, 5 days a week for 4 weeks) and normobaric oxygen controls (NBO) were analyzed by immunohistochemistry. RESULTS VEGF expression in 6-week HBO samples was elevated compared to NBO (P = .012). Staining of the 12-week HBO samples was reduced compared to 6-week HBO (P = .008) and was similar to 6- and 12-week NBO control samples. CONCLUSION HBO therapy resulted in increased VEGF expression in the defects even 2 weeks after the termination of treatment (6 weeks postsurgery).

Effects of magnesium-substituted nanohydroxyapatite coating on implant osseointegration

OBJECTIVE The objective of this study was to compare magnesium-substituted and pure hydroxyapatite coatings on the promotion of osteogenesis in vitro and on the osseointegration in vivo. METHODS Electrochemically deposited pure hydroxyapatite (EDHA) or electrochemically deposited magnesium-substituted hydroxyapatite (EDMHA) coatings were formed on the surface of pure titanium disks or implants. MC3T3-E1 preosteoblasts were cultured in the EDHA and EDMHA coated disks, and cell growth, alkaline phosphatase (ALP) activity, and osteocalcin secretion were measured at various time points. For studies on osseointegration, 30 roughened implants coated either with EDHA or EDMHA (n = 15 for each coating) were implanted in the femurs of 15 NZW rabbits. After 2, 4, and 8 weeks, femurs were retrieved and prepared for histomorphometric evaluation (n = 5 for each coating at each time point). RESULTS MC3T3-E1 cells cultured on EDMHA coated disks showed increased cell number, ALP, and osteocalcin secretion compared with the EDHA coated disks at all time points (P < 0.05 for all). Histologic observation of the coated implants showed woven bone in direct contact with both implant surfaces after 2 weeks and mature bone after 8 weeks. While there were no differences in the amount of bone between the threads at any time point, the percentage of implant in direct contact with bone (bone implant contact) was slightly higher along the EDMHA coated implants at 2 weeks (P = 0.086), although this difference was no longer seen at 4 and 8 weeks. CONCLUSION Mg-substituted HA coated surfaces promote osteogenic differentiation of preosteoblasts in vitro and may improve implant osseointegration during the early stages of bone healing compared with pure EDHA coated surfaces.

An evaluation of hydroxyapatite and biphasic calcium phosphate in combination with Pluronic F127 and BMP on bone repair.

Calcium phosphates like hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and their mixtures (biphasic calcium phosphates; BCP) are used clinically to repair bone defects. These materials can be difficult to handle and have no inherent biological activity. Handling properties of other bone substitute materials have been improved by combining them with an inert carrier such as Pluronic F-127 (Pluronic, BASF, Mt. Olive, NJ), while the addition of bone morphogenetic proteins (BMP) with such implants has also been shown to enhance bone repair. This study assessed the impact of adding Pluronic and BMPs to an HA (C-Graft™) or a BCP (80/20 HA/β-TCP ratio; Algisorb™) implants ability of promote bony repair in the rabbit calvarial defect model. Twenty-five New Zealand white rabbits were divided into 5 groups of 5 animals each. Bilateral calvarial defects were made in the parietal bones of each animal. HA or BCP alone or combined with Pluronic and/or BMP were implanted into the defect sites. Animals were euthanized at 6 weeks, postoperatively. Bone regeneration was evaluated quantitatively by histomorphometry. The amount of bone regeneration, which occurred in defects containing HA and BCP, was similar over the time period studied. Incorporating Pluronic increased handling and moldability without compromising osteoconductivity of either calcium phosphate. The addition of BMP significantly increased the amount of new bone formed with all calcium phosphates studied (P < 0.05). These results suggest that Pluronic can be added to calcium phosphates to enhance handling and moldability without any negative effects on their biocompatibility and that healing can be enhanced with the incorporation of BMPs.

Comparison of platelet-rich plasma, bovine BMP, and rhBMP-4 on bone matrix protein expression in vitro

This study investigated the potential use of platelet-rich plasma (PRP) in conjunction with mRNA expression of bone matrix proteins using bioassay and RT-PCR comparing bovine bone morphogenetic proteins (BMP), recombinant human BMP-4 (rhBMP-4) during rat bone marrow stromal cell (Mesenchymal Stem Cell) differentiation at 14 days. The results showed that all three growth factors were associated with significantly elevated alkaline phosphatase activity. PRP and bovine BMP resulted in increased protein content. The mRNA of type I collagen was expressed with all three growth factors and remained consistently elevated. Osteopontin was observed with PRP from days 1 to 7; bone sialoprotein expression was detected on days 1 and 3. PRP, bovine BMP and rhBMP-4 enhanced the steady-state expression of PDGF-A as time-dependent to day 14 and in PRP was the strongest. PTHr was expressed at days 1 and 5. Vascular endothelial growth factor expression was the most highly expressed after day 3. These findings suggest that PRP increases mRNA expression of bone matrix protein, enchances osteogenesis and angiogenesis in vitro.

Bone healing with an in situ-formed bioresorbable polyethylene glycol hydrogel membrane in rabbit calvarial defects.

OBJECTIVES The aims of this study were to test whether or not the application of an in situ-formed synthetic polyethylene glycol hydrogel (PEG) used as a biodegradable membrane for guided bone regeneration with a variety of graft materials and ambient oxygen or hyperbaric oxygen (HBO) environments would result in enhanced bone regeneration, and to observe the histologic and histomorphometric aspects of bone healing of the calvarial defects with and without a PEG membrane. STUDY DESIGN Thirty adult, skeletally mature, male New Zealand white rabbits were randomly divided into 3 groups of 10 animals each. Bilateral 15-mm-diameter critical-size defects were created in the parietal bones of each animal. Group 1 served as a control with unfilled bilateral calvarial defects, group 2 had bilateral calvarial defects filled with morcelized autogenous calvarial bone, and group 3 had bilateral calvarial defects filled with a biphasic calcium phosphate ceramic. One of the calvarial defects was randomly protected with a PEG resorbable liquid membrane in each animal. Five animals from each group underwent a course of HBO treatment (2.4 ATA 100% oxygen for 90 minutes 5 days a week for 4 weeks) and the other 5 served as control and did not receive any supplemental oxygen (normobaric). The animals were killed 6 weeks after their surgery, and their parietal bones were harvested. The specimens were analyzed with microscopic computerized tomography (microCT) scans and histomorphometrics. RESULTS The unfilled normobaric control bony defects did not heal, proving the critical-size nature of these defects. The presence of autogenous bone or bone ceramic in the defects increased the bone volume fraction and bone mineral density of the defects (P < .001). The presence of a membrane in the ungrafted and autogenous bone grafted defects resulted in a decrease in the corrected bone volume fraction (P = .002) but not in the bone ceramic grafted defects (P = .580). Bony healing of defects where the membrane was unsupported was compromised; the membrane did not maintain the desired bone regeneration volume with the unfilled and autogenous bone grafted groups. The PEG resorbable liquid membrane worked best with the bone ceramic material. HBO did not ameliorate the healing of the autogenous bone graft or ceramic filled defects in the 6-week time period of this study. CONCLUSIONS Although the PEG resorbable liquid membrane is easy to use and forms an occlusive layer, caution is recommended when using the membrane over an unsupported defect. HBO did not ameliorate bony healing with the membrane at the early 6-week time point. The authors recommend future assessment with HBO at the 12-week time point.

Effect of hyperbaric oxygen on grafted and nongrafted calvarial critical-sized defects

OBJECTIVES This study was undertaken to evaluate the effect of hyperbaric oxygen (HBO) on the repair of critical-sized defects in the presence and absence of a nonvascularized autogenous bone graft. STUDY DESIGN Ten New Zealand White rabbits were randomly divided into 2 groups of 5 animals each. Bilateral 15-mm calvarial defects were created in the parietal bones of each animal, resulting in 20 critical-sized defects. Autogenous bone grafts (ABG) were allocated to the left or right defect of each animal. Group 1 received HBO treatment at 2.4 ATA 100% oxygen for 90 minutes per day 5 days a week for 4 weeks. Group 2 served as a normobaric (NBO) control, breathing only room air. The animals in each group were humanely killed at 6 weeks. Calvaria were analyzed by micro-CT and histomorphometry. RESULTS Micro-CT analysis indicated that as expected there was a higher bone mineral density (BMD) and bone mineral content (BMC) in ABG than unfilled defects (P < .05). However, there was a significant decline in the bone mineral content (BMC) of HBO-treated grafted defects compared to NBO-treated grafted defects (P < .05). Histologically complete bridging of the defect was observed in both NBO and HBO ABG grafted defects. Histomorphometic analysis showed that HBO treatment increased new bone and marrow, and reduced fibrous tissue in the defects (P < .01 for all). Examination of residual graft showed a near significant reduction in residual graft volume (11.2 +/- 4.7 versus 19.1 +/- 7.7, HBO versus NBO P = .085) in the HBO group. The use of a graft increased new bone and marrow in the NBO group (P < .001 for both); however, in the HBO-treated animals the differences between grafted and ungrafted were not significant. CONCLUSION HBO enhances bony healing in ungrafted rabbit calvarial critical-sized defects and may increase the rate of residual graft resorption in autogenous bone-grafted defects.