Pathogenesis and treatment of a giant occipital bone defect with meningoencephalocele in an NF1 child: case report and review of the literature.

Autosomal dominantly inherited neurofibromatosis type I (NF1) is a systemic disorder caused by a mutation of a gene on chromosome 17q11.2 and characterized by multiple café-au-lait spots, lentiginous macules, Lisch nodules of the iris, and tumors of the nervous system. Bony manifestations such as scoliosis, dysplasia of the greater sphenoidal wing, tibial pseudoarthrosis, short stature, and macrocephaly have been reported in approximately 50% of patients. However, calvarial bone defects are rare. After screening 324 articles, 23 cases (12 adult and 11 pediatric patients) of occipital bone defects in NF1 patients were selected. All patients had a single/multiple bone defect over the lambdoid suture. Adjacent benign plexiform neurofibromas were observed in 14 patients (60.8%, 7 adults and 7 children); one adult patient was diagnosed with neurofibrosarcoma. Meningoencephalocele over the occipital defect was noted in 8 cases (34.78%, all adults). Cranioplasty was performed in only 17.39% of patients. Histologic examination was performed in 7 of the 15 patients with associated neurofibromas/neurofibrosarcomas. Biopsy of the bone margins surrounding the defect was performed in only one case. Pathologic examination of the herniated parieto-occipital or cerebellar tissue was not performed in any of the patients studied. We report the case of a 9-year-old girl with NF1 and a significant occipital bone defect and performed a systematic review of the relevant literature to highlight the challenges in treating this condition and to investigate the underlying mechanisms contributing to bone defects or dysplasia in NF1.

Photobiomodulation combined with adipose-derived stem cells encapsulated in methacrylated gelatin hydrogels enhances in vivo bone regeneration.

Reconstruction of bone defects is still a significant challenge. The aim of this study was to evaluate the effect of application of photobiomodulation (PBM) to enhance in vivo bone regeneration and osteogenic differentiation potential of adipose-derived stem cells (ADSCs) encapsulated in methacrylated gelatin (GEL-MA) hydrogels. Thirty-six Sprague-Dawley rats were randomly separated into 3 experimental groups (n = 12 each). The groups were control/blank defect (I), GEL-MA hydrogel (II), and ADSC-loaded GEL-MA (GEL-MA+ADSC) hydrogel (III). Biparietal critical sized bone defects (6 mm in size) are created in each animal. Half of the animals from each group (n = 6 each) were randomly selected for PBM application using polychromatic light in the near infrared region, 600-1200 nm. PBM was administered from 10 cm distance cranially in 48 h interval. The calvaria were harvested at the 20th week, and macroscopic, microtomographic, and histologic evaluation were performed for further analysis. Microtomographic evaluation demonstrated the highest result for mineralized matrix formation (MMF) in group III. PBM receiving samples of group III showed mean MMF of 79.93±3.41%, whereas the non-PBM receiving samples revealed mean MMF of 60.62±6.34 % (p=0.002). In terms of histologic evaluation of bone defect repair, the higher scores were obtained in the groups II and III when compared to the control group (2.0 for both PBM receiving and non-receiving specimens; p<0.001). ADSC-loaded microwave-induced GEL-MA hydrogels and periodic application of photobiomodulation with polychromatic light appear to have beneficial effect on bone regeneration and can stimulate ADSCs for osteogenic differentiation.

Comparison of bone regenerative capacity of donor-matched human adipose-derived and bone marrow mesenchymal stem cells.

Adipose-derived stem cells (ASC) have been used as an alternative to bone marrow mesenchymal stem cells (BMSC) for bone tissue engineering. However, the efficacy of ASC in bone regeneration in comparison with BMSC remains debatable, since inconsistent results have been reported. Comparing ASC with BMSC obtained from different individuals might contribute to this inconsistency in results. Therefore, this study aimed to compare the bone regenerative capacity of donor-matched human ASC and BMSC seeded onto poly(L-lactide-co-ε-caprolactone) scaffolds using calvarial bone defects in nude rats. First, donor-matched ASC and BMSC were seeded onto the co-polymer scaffolds to evaluate their in vitro osteogenic differentiation. Seeded scaffolds and scaffolds without cells (control) were then implanted in calvarial defects in nude rats. The expression of osteogenesis-related genes was examined after 4 weeks. Cellular activity was investigated after 4 and 12 weeks. Bone formation was evaluated radiographically and histologically after 4, 12, and 24 weeks. In vitro, ASC and BMSC demonstrated mineralization. However, BMSC showed higher alkaline phosphatase activity than ASC. In vivo, human osteogenesis-related genes Runx2 and collagen type I were expressed in defects with scaffold/cells. Defects with scaffold/BMSC had higher cellular activity than defects with scaffold/ASC. Moreover, bone formation in defects with scaffold/BMSC was greater than in defects with scaffold/ASC, especially at the early time-point. These results suggest that although ASC have the potential to regenerate bone, the rate of bone regeneration with ASC may be slower than with BMSC. Accordingly, BMSC are more suitable for bone regenerative applications.

Long-term pretreatment with alendronate inhibits calvarial defect healing in an osteoporotic rat model.

INTRODUCTION: This study aimed to observe the effects of long-term alendronate pretreatment on the healing of osteoporotic calvarial defects, and further investigate the effect of alendronate combined with once-weekly parathyroid hormone following 12 weeks of alendronate treatment in ovariectomized rats. MATERIALS AND METHODS: Thirty 3-month-old female rats were ovariectomized, and 24 rats received alendronate for 12 weeks. Then, a critical defect was created in the calvaria of all animals. Immediately after osteotomy, the animals received one of five treatments for 8 weeks: (1) continuation of vehicle (group E), (2) alendronate followed by vehicle (group A), (3) continuation of alendronate (group B), (4) alendronate followed by once-weekly parathyroid hormone alone (group C), or (5) continuation of alendronate combined with once-weekly parathyroid hormone (group D). Calvarial defect healing was assessed using dual-energy X-ray absorptiometry, micro-computed tomography, histology, and sequential fluorescence labeling. RESULTS: Group E showed a significantly higher volume of newly formed bone than groups A, B, C, and D. Evidence of new dense bone formation in group E was observed histologically. In addition, the immunohistochemical expression of runt-related transcription factor 2 was increased in group E but inhibited in groups A, B, C, and D. Sequential immunofluorescence also showed inhibited mineral apposition in groups A, B, C, and D compared with group E. CONCLUSION: The present study shows that long-term pretreatment with alendronate inhibited calvarial defect healing in osteoporotic rats, and this effect could not be reversed by stopping alendronate, switching to parathyroid hormone, or combining with once-weekly parathyroid hormone.

The immunogenic reaction and bone defect repair function of ε-poly-L-lysine (EPL)-coated nanoscale PCL/HA scaffold in rabbit calvarial bone defect.

Tissue engineering is a promising strategy for bone tissue defect reconstruction. Immunogenic reaction, which was induced by scaffolds degradation or contaminating microorganism, influence cellular activity, compromise the efficiency of tissue engineering, or eventually lead to the failure of regeneration. Inhibiting excessive immune response through modulating scaffold is critical important to promote tissue regeneration. Our previous study showed that ε-poly-L-lysine (EPL)-coated nanoscale polycaprolactone/hydroxyapatite (EPL/PCL/HA) composite scaffold has enhanced antibacterial and osteogenic properties in vitro. However, the bone defect repair function and immunogenic reaction of EPL/PCL/HA scaffolds in vivo remains unclear. In the present study, three nanoscale scaffolds (EPL/PCL/HA, PCL and PCL/HA) were transplanted into rabbit paraspinal muscle pouches, and T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17), and macrophage infiltration were analyzed after 1 week and 2 weeks to detect their immunogenic reaction. Then, the different scaffolds were transplanted into rabbit calvarial bone defect to compare the bone defect repair capacities. The results showed that EPL/PCL/HA composite scaffolds decreased pro-inflammatory Th1, Th17, and type I macrophage infiltration from 1 to 2 weeks, and increased anti-inflammatory Th2 infiltration into the regenerated area at 2 weeks in vivo, when compared to PCL and PCL/HA. In addition, EPL/PCL/HA showed an enhanced bone repair capacity compared to PCL and PCL/HA when transplanted into rabbit calvarial bone defects at both 4 and 8 weeks. Hence, our results suggest that EPL could regulate the immunogenic reaction and promote bone defect repair function of PCL/HA, which is a promising agent for tissue engineering scaffold modulation.

In vivo bone regeneration using a bioactive nanocomposite scaffold and human mesenchymal stem cells.

Due to the osteoconductive role of bioceramics, use of these bioactive nanocomposite scaffolds that can maintain their structural integrity during bone tissue repair is one of the major goals of tissue engineering. Herein, a nanofibrous poly-L-lactic acid (PLLA) scaffold was fabricated by electrospinning and then gelatin and hydroxyapatite nanoparticles (nHA) were coated over the surface of the scaffold. Osteoconductivity of the fabricated nano-composite scaffolds was then studied while grafted on the rat calvarial defects. Our results indicated that the coating of PLLA scaffold with nHA and gelatin increased the adhesion and growth of the human bone marrow derived mesenchymal stem cells (BM-MSCs) and also significantly increased the level of mineralization over a week culture period. The results of radiographic and histological studies showed that the newly created bone tissue at the defect site was significantly higher in animals treated with nanocomposite scaffolds than the empty scaffolds and control groups. This increase in the defect reconstruction was significantly increased after culturing BM-MSCs on the scaffolds, especially nanocomposite scaffolds. It can be concluded that the combination of nanocomposite scaffolds and BM-MSCs could be a very good candidate for treatment of bone lesions and could be considered as a bony bioimplant.

In Vivo Efficacy of Neutrophil-Mediated Bone Regeneration Using a Rabbit Calvarial Defect Model.

Reconstruction of bone due to surgical removal or disease-related bony defects is a clinical challenge. It is known that the immune system exerts positive immunomodulatory effects on tissue repair and regeneration. In this study, we evaluated the in vivo efficacy of autologous neutrophils on bone regeneration using a rabbit calvarial defect model. Methods: Twelve rabbits, each with two surgically created calvarial bone defects (10 mm diameter), were randomly divided into two groups; (i) single application of neutrophils (SA-NP) vs. SA-NP control, and (ii) repetitive application of neutrophils (RA-NP) vs. RA-NP control. The animals were euthanized at 4 and 8 weeks post-operatively and the treatment outcomes were evaluated by micro-computed tomography, histology, and histomorphometric analyses. Results: The micro-CT analysis showed a significantly higher bone volume fraction (bone volume/total volume) in the neutrophil-treated groups, i.e., median interquartile range (IQR) SA-NP (18) and RA-NP (24), compared with the untreated controls, i.e., SA-NP (7) and RA-NP (14) at 4 weeks (p < 0.05). Similarly, new bone area fraction (bone area/total area) was significantly higher in neutrophil-treated groups at 4 weeks (p < 0.05). Both SA-NP and RA-NP had a considerably higher bone volume and bone area at 8 weeks, although the difference was not statistically significant. In addition, immunohistochemical analysis at 8 weeks revealed a higher expression of osteocalcin in both SA-NP and RA-NP groups. Conclusions: The present study provides first hand evidence that autologous neutrophils may have a positive effect on promoting new bone formation. Future studies should be performed with a larger sample size in non-human primate models. If proven feasible, this new promising strategy could bring clinical benefits for bone defects to the field of oral and maxillofacial surgery.

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model.

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.

Giant skull vault defect in a child caused by Langerhans cell histiocytosis: the "calvaria-eating disease".

Langerhans cell histiocytosis (LCH) is a rare disease of young children that can be difficult to diagnose and treat. Clinical manifestations can sometimes be quite unusual, and bony lesions can reach a large size before treatment is sought. We present a unique case of a 3-year-old male child with a painless giant calvarial defect and cystic swelling. Complete removal of the cyst with curettage of the involved bone margins and cranioplasty was done using bone cement (polymethyl methacrylate, PMMA). However, there was a flare up of the disease due to abnormal inflammatory reaction to PMMA, and cranioplasty bone flap had to be removed. The child did well after PMMA bone flap removal.LCH should be suspected in a male child presenting with a large calvarial defect and no history of trauma. The use of materials, viz., hydroxyapatite, poly-DL-lactide (PDLLA), and PMMA for cranioplasty in LCH, may cause reactivation of the disease process and therefore must be used cautiously in these patients.

Local supplementation with plant-derived recombinant human FGF2 protein enhances bone formation in critical-sized calvarial defects.

Numerous studies have demonstrated the advantages of plant cell suspension culture systems in producing bioactive recombinant human growth factors. This study investigated the biological activity of recombinant basic human fibroblast growth factor (rhFGF2) protein produced by a plant culture system to enhance new bone formation in a bone defect mouse model. The human FGF2 cDNA gene was cloned into a plant expression vector driven by the rice α-amylase 3D promoter. The vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), and the clone with the highest expression of rhFGF2 was selected. Maximum accumulation of rhFGF2 protein (approximately 28 mg/l) was reached at 13 day post-incubation. Male C57BL/6 mice underwent calvarial defect surgery and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 5 µg of plant-derived rhFGF2 (p-FGF2) protein or E. coli-derived rhFGF2 (e-FGF2) protein. Similar to the effects of e-FGF2, local delivery with p-FGF2 enhanced bone healing in the damaged region to higher levels than the ACS group. Exogenous addition of p-FGF2 or e-FGF2 exhibited similar effects on proliferation, mineralization, and osteogenic marker expression in MC3T3-E1 cells. Together, the current findings support the usefulness of this plant-based expression system for the production of biologically active rhFGF2.

Synergistic effect of hyperbaric oxygen therapy with PTH [1-34] on calvarial bone graft in irradiated rat.

OBJECTIVE: To determine the synergistic effect of parathyroid hormone (PTH) [1-34] in combination with hyperbaric oxygen (HBO) on bone graft in a rat calvarial bone defect model under impaired osteogenic conditions. MATERIALS AND METHODS: Twenty-four rats were divided into three groups. Localized radiation with a single 12 Gy dose was administered to the calvaria. Four weeks after radiation, calvarial circular defects were created in the parietal bones. All defects were filled with biphasic calcium phosphate. After the bone graft, PTH [1-34] was injected subcutaneously, and HBO was administered. At 6 weeks after the bone graft, the rats were sacrificed, and specimens were harvested. RESULTS: Histomorphometric evaluation showed that the percentage of new bone area was higher in the PTH and PTH/HBO groups than in the control group. The percent residual material area was decreased in the PTH/HBO group compared with the control group. The percentage blood vessel number was highest in the PTH group. Micro-CT evaluation showed that the new bone volume was highest in the PTH/HBO group. The residual material volume was lowest in the PTH/HBO group. CONCLUSION: Within the limitations of this study, our data indicate that PTH combined with HBO may reverse radiation-induced impairment of bone healing.

In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells.

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties.

Comparing the Osteogenic Potentials and Bone Regeneration Capacities of Bone Marrow and Dental Pulp Mesenchymal Stem Cells in a Rabbit Calvarial Bone Defect Model.

The bone regeneration efficiency of bone marrow mesenchymal stem cells (BMSCs) and dental pulp mesenchymal stem cells (DPSCs) combined with xenografts in the craniofacial region remains unclear. Accordingly, this study commenced by comparing the cell morphology, cell proliferation, trilineage differentiation, mineral synthesis, and osteogenic gene expression of BMSCs and DPSCs in vitro. Four experimental groups (empty control, Bio-Oss only, Bio-Oss+BMSCs, and Bio-Oss+DPSCs) were then designed and implanted in rabbit calvarial defects. The BMSCs and DPSCs showed a similar morphology, proliferative ability, surface marker profile, and trilineage-differentiation potential in vitro. However, the BMSCs exhibited a higher mineral deposition and expression levels of osteogenic marker genes, including alkaline phosphatase (ALP), runt related transcription factor 2 (RUNX2), and osteocalcin (OCN). In the in vivo studies, the bone volume density in both MSC groups was significantly greater than that in the empty control or Bio-Oss only group. Moreover, the new bone formation and Collagen I / osteoprotegerin protein expressions of the scaffold+MSC groups were higher than those of the Bio-Oss only group. Finally, the Bio-Oss+BMSC and Bio-Oss+DPSC groups had a similar bone mineral density, new bone formation, and osteogenesis-related protein expression. Overall, the DPSCs seeded on Bio-Oss matched the bone regeneration efficacy of BMSCs in vivo and hence appear to be a promising strategy for craniofacial defect repair in future clinical applications.

Treatment of calvarial defects by resorbable and non-resorbable sonic activated polymer pins and mouldable titanium mesh in two dogs: a case report.

BACKGROUND: To date, calvarial defects in dogs have traditionally been addressed with different types of implants including bone allograft, polymethylmethacrylate and titanium mesh secured with conventional metallic fixation methods. This report describes the use of an absorbable and non absorbable novel polymer fixation method, Bonewelding® technology, in combination with titanium mesh for the repair of calvarial defects in two dogs. The clinical outcomes and comparative complication using resorbable and non-resorbable thermoplastic pins were compared. CASE PRESENTATION: This report of two cases documents the repair of a traumatic calvarial fracture in an adult male Greyhound and a cranioplasty following frontal bone tumor resection in an adult female Cavalier King Charles Spaniel with the use of a commercially available titanium mesh secured with an innovative thermoplastic polymer screw system (Bonewelding®). The treatment combination aimed to restore cranial structure, sinus integrity and cosmetic appearance. A mouldable titanium mesh was cut to fit the bone defect of the frontal bone and secured with either resorbable or non-resorbable polymer pins using Bonewelding® technology. Gentamycin-impregnated collagen sponge was used intraoperatively to assist with sealing of the frontal sinuses. Calvarial fracture and post-operative implant positioning were advised using computed tomography. A satisfactory restoration of skull integrity and cosmetic result was achieved, and long term clinical outcome was deemed clinically adequate with good patient quality of life. Postoperative complications including rostral mesh uplift with minor associated clinical signs were encountered when resorbable pins were used. No postoperative complications were experienced in non-resorbable pins at 7 months follow-up, by contrast mesh uplift was noted 3 weeks post-procedure in the case treated using absorbable pins. CONCLUSIONS: The report demonstrates the innovative use of sonic-activated polymer pins (Bonewelding® technology) alongside titanium mesh is a suitable alternative technique for skull defect repair in dogs. The use of Bonewelding® may offer advantages in reduction of surgical time. Further, ultrasonic pin application may be less invasive than alternative metallic fixation and potentially reduces bone trauma. Polymer systems may offer enhanced mesh-bone integration when compared to traditional metallic implants. The use of polymer pins demonstrates initial potential as a fixation method in cranioplasty. Initial findings in a single case comparison indicate a possible advantage in the use of non-absorbable over the absorbable systems to circumvent complications associated with variable polymer degradation, further long term studies with higher patient numbers are required before reliable conclusions can be made.

Effects of Enhanced Hydrophilic Titanium Dioxide-Coated Hydroxyapatite on Bone Regeneration in Rabbit Calvarial Defects.

The regeneration of bone defects caused by periodontal disease or trauma is an important goal. Porous hydroxyapatite (HA) is an osteoconductive graft material. However, the hydrophobic properties of HA can be a disadvantage in the initial healing process. HA can be coated with TiO2 to improve its hydrophilicity, and ultraviolet irradiation (UV) can further increase the hydrophilicity by photofunctionalization. This study was designed to evaluate the effect of 5% TiO2-coated HA on rabbit calvarial defects and compare it with that of photofunctionalization on new bone in the early stage. The following four study groups were established, negative control, HA, TiO2-coated HA, and TiO2-coated HA with UV. The animals were sacrificed and the defects were assessed by radiography as well as histologic and histomorphometric analyses. At 2 and 8 weeks postoperatively, the TiO2-coated HA with UV group and TiO2-coated HA group showed significantly higher percentages of new bone than the control group (p < 0.05). UV irradiation increased the extent of new bone formation, and there was a significant difference between the TiO2-coated HA group and TiO2-coated HA with UV group. The combination of TiO2/HA and UV irradiation in bone regeneration appears to induce a favorable response.

The inhibitory effect of zoledronate on early-stage osteoinduction by recombinant human bone morphogenetic protein 2 in an osteoporosis model.

This study evaluated the effect of the combined treatment of intravenous zoledronic acid (ZA, 0.08 mg/kg) and rhBMP-2 (5 µg) on osteogenesis in a calvarial defect model of ovariectomized SD rats. New bone formation was evaluated 4 or 8 weeks after calvarial defect implantation using micro-CT and histology. Micro-CT results revealed that the rhBMP-2 group showed significantly higher calvarial defect coverage ratio compared with the ZA + rhBMP-2 group at 4 weeks. In addition, bone formation indices were significantly lower in ZA + rhBMP-2 group when compared with the rhBMP-2 group after 4 weeks, which indicates a negative effect of ZA on the initial bone formation and the bone quality. At 8 weeks, the negative effect induced by ZA treatment was alleviated as time passed. Histological examination showed similar results to the micro-CT measurements. In conclusion, although ZA treatment lowered the new bone formation induced by rhBMP-2 initially, as time passed, the negative effect was decreased.

Novel bioresorbable strontium hydroxyapatite membrane for guided bone regeneration.

OBJECTIVES: Membrane materials have been widely used for guided bone regeneration (GBR). However, due to bio-functional limitation of the current membranes, the ideal resorbable membrane that can stimulate bone regeneration has yet to be developed. This study seeks to investigate the effects of a strontium hydroxyapatite (SrHA)-containing membrane for GBR. MATERIAL AND METHODS: Strontium hydroxyapatite powder was synthesized and mixed with gelatin solution to the final concentration of 10 mg/ml (Sr10) and 20 mg/ml (Sr20). Approximately 100-µm-thick membranes were fabricated, and the mechanical properties and strontium ion release pattern were analyzed. Rat bone marrow stromal cell (BMSC) responses were investigated in vitro. Bilaterial rat calvarial defects were used in vivo to compare the SrHA membranes against commercially available collagen membranes and evaluated radiologically and histologically. RESULTS: Strontium hydroxyapatite membranes exhibited higher elasticity and strength than the collagen membrane, and slow strontium ion release was also confirmed. No BMSC cytotoxicity was found on the SrHA membranes, and the alkaline phosphatase positively stained area was significantly greater than the collagen membrane at earlier time point. At 4 weeks, both micro-CT and histological analyses revealed that the Sr20 group yielded significantly greater bone formation. CONCLUSIONS: The SrHA-containing membrane developed in this study was found to be a biocompatible material that can stimulate BMSC differentiation as well as bone regeneration and maturation in rat calvarial defects at early time point compared with collagen membrane. The best result was observed in Sr20 group, which can be potentially effective for GBR.

Enhanced osteogenicity of the demineralized bone-dermal matrix composite by the optimal partial demineralization for sustained release of bioactive molecules.

Allogenic demineralized bone matrix (DBM), processed to expose bioactive proteins imbedded by calcium salts, is widely used for bone repair and regeneration as an alternative to the autologous bone graft. However, demineralized bone matrices from tissue banks vary significantly in residual calcium content and osteogenicity for clinical bone regeneration. The present study produced DBM with various residual calcium contents by partial demineralization using ethylenediaminetetraacetic acid disodium (EDTA) and hydrochloric acid. Compositional analysis reveals that, as the percent weight loss of bone materials increases from 0% to 74.9% during demineralization, the residual calcium content of DBM decreases from 24.8% to 0.2% and collagen content increases from 29.7% to 92.6%. Calorimetrical analysis and Fourier transform infrared (FTIR) analysis demonstrated that demineralization to the residual calcium content of <4% enables the complete exposure and/or release of bone collagen fibers and other bioactive molecules. In order to evaluate the relationship between the extent of demineralization and the osteogenicity of DBM, DBM particles were fabricated with the aid of acellular dermal matrix (ADM) microfibers to form flexible foam-like DBM/ADM composites. Proteomic analysis identified various type collagens and bone formation-related bioactive molecules in both ADM and DBM. Using the rat bilateral Φ = 5 mm calvarium defect repair model, the study had shown that the DBM/ADM composite with ~20% DBM residual calcium (e.g., ~40% calcium being removed) maximized the osteogenicity for bone defect repair after 4 and 8 weeks. DBM with ~40% calcium removal had the maximal osteogenicity presumably through the sustained release of bioactive molecules during the process of bone regeneration.

3D printed polylactic acid-based nanocomposite scaffold stuffed with microporous simvastatin-loaded polyelectrolyte for craniofacial reconstruction.

Critical sized craniofacial defects are among the most challenging bone defects to repair, due to the anatomical complexity and aesthetic importance. In this study, a polylactic acid/hardystonite-graphene oxide (PLA/HTGO) scaffold was fabricated through 3D printing. In order to upgrade the 3D printed scaffold to a highly porous scaffold, its channels were filled with pectin-quaternized chitosan (Pec-QCs) polyelectrolyte solution containing 0 or 20 mg/mL of simvastatin (Sim) and then freeze-dried. These scaffolds were named FD and FD-Sim, respectively. Also, similar PLA/HTGO scaffolds were prepared and dip coated with Pec-QCs solution containing 0 or 20 mg/mL of Sim and were named DC and DC-Sim, respectively. The formation of macro/microporous structure was confirmed by morphological investigations. The release of Sim from DC-Sim and FD-Sim scaffolds after 28 days was measured as 77.40 ± 5.25 and 86.02 ± 3.63 %, respectively. Cytocompatibility assessments showed that MG-63 cells had the highest proliferation, attachment and spread on the Sim containing scaffolds, especially FD-Sim. In vivo studies on a rat calvarial defect model revealed that an almost complete recovery occurred in the group treated with FD-Sim scaffold after 8 weeks and the defect was filled with newly formed bone. The results of this study acknowledge that the FD-Sim scaffold can be a perfect candidate for calvarial defect repair.