Greater Bone Formation Research Articles

A histological examination of spinal reconstruction using a frozen bone autograft.

Our aim was to compare the process of bone formation after reconstruction of the vertebral body using a titanium cage with either a liquid nitrogen-treated (frozen) bone autograft or non-treated fresh bone autograft. Twelve canine beagles underwent anterior reconstruction of the 5th lumbar vertebrae using a titanium cage and bone autograft. Bone formation was compared across four experimental groups: fresh bone autograft groups, with animals sacrificed at either 8 or 16 weeks post-reconstruction, and liquid nitrogen-treated (frozen) bone autograft groups, with animals again sacrificed at either 8 or 16 weeks post-reconstruction. Bone formation was evaluated histologically by calculating the proportion of ‘reaction’ and ‘mature bone’ regions at the ends of the cage, its center, and ventral/dorsal sides. The reaction region contained osteocytes with a nucleus and osteoblasts accumulated on the surface of an osteoid, while a laminar structure was visible for mature bone regions. For fresh bone autografts, the reaction and mature bone regions significantly increased from 8 to 16 weeks post-reconstruction. By comparison, for frozen autografts, the reaction bone region did not significantly increase from 8 to 16 weeks post-reconstruction, while the mature bone region did increase over this time period. The proportion of reaction bone was higher at the ends and dorsal side of the cage at 8 weeks, for both graft types, with greater bone formation at the center of the cage at 16 weeks only for the fresh bone autograft. Therefore, bone formation in the anterior spinal reconstruction site tended to be delayed when using a frozen bone autograft compared to a fresh bone autograft. The bone formation process, however, was similar for both groups, beginning at the ends and dorsal side of the cage adjacent to the surrounding vertebral bone.

Effect of a Rapidly Resorbable Calcium Alkali Phosphate Bone Grafting Material on Osteogenesis after Sinus Floor Augmentation in Humans

Sinus floor augmentation (SFA) has become a well-established pre-implantology procedure for alveolar ridge augmentation of the posterior maxilla. Using bioceramic bone substitutes avoids second-site surgery for autograft harvesting. Compared to the bone substitutes which are currently clinically available, there is a significant need for bone substitutes which degrade more rapidly, but still stimulate osteogenesis at the same time. This has led to the development of bioactive, rapidly resorbable calcium alkali orthophosphate (CAOP) materials, which have a greater solubility than tricalcium phosphate. In this study the biodegradability and effect of a silica containing CAOP (Si-CAOP) on osteogenesis was evaluated in human biopsies sampled 6 months after SFA and compared to that of TCP utilizing hard tissue histology, histomorphometry and immunohistochemical analysis of osteogenic marker expression. Both materials facilitated bone formation and matrix mineralization, which were still actively progressing from the sinus floor in an apical direction 6 months after SFA. With the Si-CAOP grafting material however, bone formation, the bone-biomaterial-contact, i.e. bone-bonding, and particle degradation were significantly greater compared to TCP in the apical region of the biopsies, i.e. at the largest distance from the native bone of the sinus floor. This was accompanied by greater expression of Col I, BSP and OC in the newly formed bone tissue in the Si-CAP samples compared to TCP. Six months after implantation Si-CAOP facilitated greater bone formation and biodegradability than the TCP graft material, whose excellent osteoconductive properties have been widely documented. Consequently, Si-CAOP can be regarded as excellent grafting material for SFA in a clinical setting.

Recombinant human IGF-1 produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects

Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). 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 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration.

Effect of sex-hormone levels, sex, body mass index and other host factors on human craniofacial bone regeneration with bioactive tricalcium phosphate grafts

Little is known regarding the associations between sex-hormone levels, sex, body mass index (BMI), age, other host factors and biomaterial stimulated bone regeneration in the human craniofacial skeleton. The aim of this study was to elucidate the associations between these factors and bone formation after sinus floor augmentation procedures (SFA) utilizing a bioactive tricalcium phosphate (TCP) bone grafting material. We conducted a prospective study in a human population in which 60 male and 60 female participants underwent SFA and dental implant placement using a staged approach. BMI as well as levels of serum estradiol (E2), total testosterone (TT), and the free androgen index (FAI) were measured by radioimmunoassay and electrochemoluminescent-immunoassay. At implant placement, 6 months after SFA, bone biopsy specimens were harvested for hard tissue histology, the amount of bone formation was evaluated by histomorphometry and immunohistochemical analysis of osteogenic marker expression. The Wilcoxon rank-sum U test, Spearman correlations and linear regression analysis were used to explore the association between bone formation and BMI, hormonal and other host factors. BMI and log E2 were significantly positively associated with bone formation in male individuals (p<0.05). Histomorphometry revealed trends toward greater bone formation and osteogenic marker expression with non-smokers compared to smokers. In male patients, higher E2 levels and higher BMI enhanced TCP stimulated craniofacial i.e. intramembranous bone repair.

The influence of collagen membrane and autogenous bone chips on bone augmentation in the anterior maxilla: a preclinical study.

To evaluate the effect of a resorbable collagen membrane and autogenous bone chips combined with deproteinized bovine bone mineral (DBBM) on the healing of buccal dehiscence-type defects. The second incisors and the first premolars were extracted in the maxilla of eight mongrels. Reduced diameter, bone-level implants were placed 5weeks later. Standardized buccal dehiscence-type defects were created and grafted at implant surgery. According to an allocation algorithm, the graft composition of each of the four maxillary sites was DBBM+membrane (group D+M), autogenous bone chips+DBBM+membrane (group A+D+M), DBBM alone (group D) or autogenous bone chips+DBBM (group A+D). Four animals were sacrificed after 3weeks of healing and four animals after 12weeks. Histological and histomorphometric analyses were performed on oro-facial sections. The pattern of bone formation and resorption within the grafted area showed high variability among the same group and healing time. The histomorphometric analysis of the 3-week specimens showed a positive effect of autogenous bone chips on both implant osseointegration and bone formation into the grafted region (P<0.05). The presence of the collagen membrane correlated with greater bone formation around the DBBM particles and greater bone formation in the grafted region after 12weeks of healing (P<0.05). The oro-facial width of the augmented region at the level of the implant shoulder was significantly reduced in cases where damage of the protection splints occurred in the first week of healing (P<0.05). The addition of autogenous bone chips and the presence of the collagen membrane increased bone formation around DBBM particles. Wound protection from mechanical noxa during early healing may be critical for bone formation within the grafted area.

Titanium scaffold osteogenesis in healthy and osteoporotic rats is improved by the use of low-level laser therapy (GaAlAs).

The present study aimed to assess the effects of low-level laser therapy (GaAlAs) on the bone repair process within titanium scaffolds in the femurs of healthy and osteoporotic rats. Fifty-six rats were divided into four groups: group Sh: SHAM animals that received scaffolds; group LSh: SHAM animals that received scaffolds and were subjected to laser therapy; group OV: ovarietomized (OVX) animals that received scaffolds; and group LOV: OVX animals that received scaffolds and were subjected to laser therapy. Thirty days following ovariectomy or sham surgery, scaffolds were implanted in the left femurs of all animals in the study. Immediately after opening the surgical site, the inner part of the surgical cavity was stimulated with low-level laser (GaAlAs). In addition to this procedure, the laser group was also subjected to sessions of low-level laser therapy (LLLT) at 48-h intervals, with the first session performed immediately after surgery. The rats were sacrificed at 2 and 6weeks, time in which femur fragments were submitted for histological and histomorphometric examination, and skin tissue above the scaffold was submitted to histological analysis. At the end of the study, greater bone formation was observed in the animals submitted to LLLT. At 2 and 6weeks, statistically significant differences were observed between LSh and Sh groups (p = 0.009 and 0.0001) and LOV and OV (p = 0.0001 and 0.0001), respectively. No statistical difference was observed when assessing the estrogen variable. On the basis of our methodology and results, we conclude that LLLT improves and accelerates bone repair within titanium scaffolds in both ovariectomized and healthy rats, when compared to animals not subjected to radiation.

Comparative study of recombinant human bone morphogenetic protein-2 carriers in rat subcutaneous tissues: Pilot study

The purpose of this study was to evaluate and compare the potential of using various biomaterials as a carrier of rhBMP-2 in the subcutaneous layer of rats. 5 subcutaneous defects were made in each of the dorsal surfaces of 10 rats. Then, the rats were divided into the following groups: Control group: sham-surgery control, Experimental group 1: rhBMP-2/Bio-Oss®, Experimental group 2: rhBMP-2/Collatape®, Experimental group 3: rhBMP-2/nCS. Histological and histometrical analysis were performed after the rats were sacrificed at 2 and 4 weeks. Regardless of the type of carrier, the groups treated with rhBMP-2 showed significantly greater bone formation when compared to the control group. The amount of new bone formation was greater at 4 week than 2 week. Total bone formation (%) at 4 week was the greatest in rhBMP/Bio-Oss® (26.76±10.13) group, followed by rhBMP/Collatape® (20.38±5.27) and rhBMP/nCS (4.34±2.58). Within the limitation of this study, the results showed that Bio-Oss®, Collatape®, nanocalcium sulfate all showed new bone formation when used as a carrier of rhBMP-2 in the subcutaneous layer of rats, indicating potential use as carriers for rhBMP-2.

Serum sclerostin and DKK1 in relation to exercise against bone loss in experimental bed rest.

The impact of effective exercise against bone loss during experimental bed rest appears to be associated with increases in bone formation rather than reductions of bone resorption. Sclerostin and dickkopf-1 are important inhibitors of osteoblast activity. We hypothesized that exercise in bed rest would prevent increases in sclerostin and dickkopf-1. Twenty-four male subjects performed resistive vibration exercise (RVE; n=7), resistive exercise only (RE; n=8), or no exercise (control n=9) during 60 days of bed rest (2nd Berlin BedRest Study). We measured serum levels of BAP, CTX-I, iPTH, calcium, sclerostin, and dickkopf-1 at 16 time-points during and up to 1year after bed rest. In inactive control, after an initial increase in both BAP and CTX-I, sclerostin increased. BAP then returned to baseline levels, and CTX-I continued to increase. In RVE and RE, BAP increased more than control in bed rest (p≤0.029). Increases of CTX-I in RE and RVE did not differ significantly to inactive control. RE may have attenuated increases in sclerostin and dickkopf-1, but this was not statistically significant. In RVE there was no evidence for any impact on sclerostin and dickkopf-1 changes. Long-term recovery of bone was also measured and 6-24 months after bed rest, and proximal femur bone mineral content was still greater in RVE than control (p=0.01). The results, while showing that exercise against bone loss in experimental bed rest results in greater bone formation, could not provide evidence that exercise impeded the rise in serum sclerostin and dickkopf-1 levels.

Avaliação histomorfométrica da associação entre biovidro e osso bovino liofilizado no tratamento de defeitos ósseos críticos criados em calvárias de ratos. Estudo piloto

OBJETIVO: Avaliar histomorfometricamente o efeito de biovidro (B), osso bovino liofilizado (OB) ou da mistura desses dois biomateriais (B/OB - 1:1) no reparo de defeitos ósseos críticos em calvária de ratos. MATERIAL E MÉTODO: Defeitos ósseos (8 mm Ø) foram criados cirurgicamente na calvária de 24 ratos, distribuídos em 4 grupos com 6 animais, de acordo com o tipo de biomaterial: coágulo sanguíneo (GC), biovidro (GB), osso bovino liofilizado (GOB) e a mistura desses dois biomateriais (GB/OB). Os animais foram eutanasiados após 15 e 60 dias do procedimento cirúrgico (3 animais por período). A avaliação histológica foi baseada na descrição da morfologia dos tecidos neoformados, enquanto para a avaliação histomorfométrica foi realizada quantificação da porcentagem de tecido ósseo, de tecido conjuntivo fibroso neoformados e de biomaterial remanescente no defeito ósseo. RESULTADO: Nos dois períodos experimentais, a análise histológica apresentou neoformação óssea, principalmente nas bordas dos defeitos, e ao redor de partículas de biomateriais remanescentes. A avaliação histomorfométrica demonstrou que no período de 15 dias o grupo GC apresentou maior percentagem de tecido ósseo em relação aos demais grupos estudados, enquanto que aos 60 dias o grupo GOB apresentou maior porcentagem de tecido ósseo em relação ao grupo GB. CONCLUSÃO: O osso bovino liofilizado apresentou maior formação óssea em relação ao biovidro, mas nenhum dos biomateriais foi superior ao coágulo. A associação do biovidro e osso bovino liofilizado não adicionou vantagem à formação óssea.

Effect of Er,Cr:YSGG laser application in the treatment of experimental periodontitis

The purpose of this study was to evaluate the influence of an erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser in the absence or presence of manual scaling and root planning (SRP) for the treatment of induced periodontitis in rats. Ligatures were placed in the subgingival region of the maxillary first molar. After a 7-day period, the ligatures were removed, and 40 rats were randomly divided into four groups (G), as follows: (GI) no treatment, (GII) scaling and root planning (SRP) with curettes, (GIII) Er,Cr:YSGG laser irradiation and (GIV) SRP with curettes followed by Er,Cr:YSGG laser irradiation. Seven and 30 days after the treatment, the animals were sacrificed and histologic, histometric and immunohistochemistry analyses were performed. All groups showed similar histopathological characteristics during the evaluation period. The histometric analysis was confirmed using Bonferroni and paired t tests. At 7 and 30 days, groups II, III and IV exhibited greater bone formation in the furcation area when compared to group I (p < 0.0001; p < 0.05). During the 7-day period, the groups irradiated with the laser (III and IV) showed a statistically larger new bone area than the group treated with SRP (II) (p < 0.01). Immunohistochemistry analysis revealed that the control group exhibited a higher expression of tartrate-resistant acid phosphatase (TRAP) and the receptor activator of nuclear factor κΒ ligand (RANKL) when compared to groups II, III and IV (p < 0.05). All treatments were able to reduce the inflammatory processes, consequently enabling the repair of periodontal tissues. The results achieved with the application of the Er,Cr:YSGG laser suggest that this laser can stimulate greater bone formation, especially over a shorter period of time.

Performance of β-tricalcium phosphate granules and putty, bone grafting materials after bilateral sinus floor augmentation in humans

Sinus floor augmentation (SFA) using bone grafting materials, and in particular calcium phosphates (CaP), is a well-established pre-implantology procedure. The use of CaP simplifies SFA procedures. β-tricalcium phosphate (β-TCP) is amply used for SFA. This study evaluated the clinical and osteogenic performance of β-TCP granules (TCP-G) and a β-TCP putty (TCP-P) bone graft material. TCP-P consisted of TCP-G in a hyaluronic acid (HyA) carrier. Bone formation, volume stability and osteogenic marker expression after bilateral SFA in patients was assessed. Eight patients were selected for a split-mouth design. Biopsies obtained six months after SFA, were processed for immunohistochemical analysis of collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Histomorphometric analysis determined bone, grafting material and marrow space percentages. Cone-beam computed tomography was used to calculate the graft volume and its stability. Both materials allowed excellent bone regeneration and volume stability. TCP-P displayed better surgical handling properties, greater bone formation, higher expression of Col I, ALP, OC and BSP; as well as significantly lower grafting volume reduction values. HyA had no adverse effect on TCP-P performance. Due to its clinical and osteogenic performance, TCP-P can be regarded as excellent bone grafting material for SFA.

Postextraction ridge preservation and augmentation with mineralized allograft with or without recombinant human platelet-derived growth factor BB (rhPDGF-BB): a consecutive case series.

In an attempt to reduce postextraction alveolar bone resorption, ridge preservation and augmentation procedures have become standard-of-care treatment following tooth removal. This consecutive case series compares histologic and histomorphometric bone regenerative findings at 4 months following grafting for ridge preservation and augmentation in intact sockets and sockets with buccal wall defects. Sites were treated with mineralized allograft alone (control) or in combination with 0.3 mg/mL recombinant human platelet-derived growth factor BB (rhPDGF-BB) (test). Sites were allowed to heal for 4 months and then re-entered for trephine core biopsy and implant placement. At the end of 4 months, the mean percent remaining mineralized allograft was statistically significantly less in the test group than in the control group. The difference in mean percent vital bone between the groups showed a strong trend toward greater bone formation for the test group (41.8%) compared to the control group (32.5%) at the end of 4 months. Addition of growth factor signaling molecules to current grafting procedures may lead to accelerated bone regeneration, making it possible to successfully place implants at earlier time points.

Secreted phosphoprotein 24 kD (Spp24) and Spp14 affect TGF-β induced bone formation differently.

Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) have opposing but complementary functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore, regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal truncation products all bind to TGF-β1 and TGF-β2 with a similar but somewhat less affinity than they bind BMP-2; that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-β with greater affinity than do the truncation products; that FL-Spp24 inhibits TGF-β2 induced bone formation in vivo, but Spp14.5 does not; and that co-administration of FL-Spp24 or Spp14.5 with TGF-β2 in vivo is associated with a reduction in the amount of cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-dose TGF-β administration in vivo is associated with greater bone formation than high-dose TGF-β administration, and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-β activity or availability during bone growth and development. The similarities and differences of the interactions between Spp24 proteins and TGF-β compared to the interaction of the Spp24 proteins and BMPs have significant implications with respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal disorders.

Experimental Drug Adds Bone After Bisphosphonate Treatment

MINNEAPOLIS – Odanacatib added bone density in elderly postmenopausal women with osteoporosis previously treated with alendronate, according to a phase II trial involving 243 patients. The investigational oral cathepsin K inhibitor began to distinguish itself from placebo at 12 months. At 2 years, the change from baseline in femoral neck bone mineral density (BMD) was increased by 1.73%, compared with a loss of 0.94% for placebo (P < .001), researchers reported at the annual meeting of the American Society for Bone and Mineral Research. BMD changes in the treatment group at 2 years were also significantly different from placebo at the trochanter (1.83% vs. –1.35%) and for the total hip (0.83% vs. –1.87%, both P < .001). All of the women studied had received alendronate (Fosamax) for at least 3 years. Patients could have been off the bisphosphonate for up to 3 months prior to enrollment, but many switched directly over to odanacatib, said study coauthor Dr. Albert Leung, executive director of clinical research at Merck Research Laboratories. “This drug has the potential to give additional benefits when [patients] have been treated with alendronate for a number of years and the treatment effect has reached a plateau,” he said in an interview. In July 2012, a pivotal 16,731-patient, phase III trial of odanacatib was stopped after an interim analysis showed a “favorable benefit-risk profile” for fracture reduction in postmenopausal women with previously untreated osteoporosis, Dr. Leung noted. The study's data monitoring committee, however, flagged safety concerns in “certain selected areas” for further follow-up. Those safety risks have not been identified as the trial is still closing, but will be monitored along with efficacy in a double-blind, placebo-controlled trial of 5 years of treatment, he said. Merck, which plans to submit regulatory applications for odanacatib in the United States and Europe in the first half of 2013, has high hopes for the drug because of its novel mechanism of action. Odanacatib inhibits cathepsin K, the primary protease in osteoclasts that breaks down bone collagen during bone resorption. Unlike traditional antiresorptive drugs like bisphosphonates, however, odanacatib does not interfere with the function of the entire osteoclast or reduce the number of osteoclasts. This characteristic is important, as osteoclasts secrete signaling factors to stimulate osteoblasts, the cells responsible for bone formation. As a result, there is greater bone formation with odanacatib, Dr. Leung explained. The phase II trial enrolled women at least 60 years of age (mean 71 years) with a BMD T score of –2.5 to more than –3.5 at any hip site without a prior fragility fracture or women with a history of fragility fracture (except hip fracture) and a BMD T score of –1.5 and more than –3.5 at any hip site. The women were randomly assigned to odanacatib 50 mg once weekly or placebo for 24 months, as well as 5,600 IU of vitamin D per week and calcium at dosages up to 1,200 mg/day. The study was not powered to assess fractures. At 24 months, the change in BMD at the lumbar spine was 2.28% for odanacatib vs. a loss of 0.30% with placebo (P < .001). BMD change was not significant at the distal forearm. A biomarker of bone resorption increased with placebo but decreased with odanacatib. A bone-formation marker rose both with placebo and odanacatib, but the gain was more with the drug. Most unexpected, however, was an increase in a resorption marker with once-weekly odanacatib. Dr. Leung said the finding appears to correlate with the bone density changes because the marker's levels remained relatively stable during the first 12 months of treatment before rising in the second year. Finally, adverse events were similar in both groups. The most common adverse events in the odanacatib and placebo arms were urinary tract infection (11.5% vs. 16.5%, respectively), back pain (11.5% vs. 9.9%), arthralgia (9% vs. 9.9%), and fractures (4.9% vs. 13.2%). Treatment discontinuation rates due to adverse events were 9% vs. 3.3%, he said. Dr. Leung and several of his coauthors are employees of Merck, the trial sponsor.

The Potential Use of Allogeneic Platelet-Rich Plasma for Large Bone Defect Treatment: Immunogenicity and Defect Healing Efficacy

Autologous platelet-rich plasma (PRP) has been extensively investigated for large bone defect treatment, but its clinical application is harassed by controversial outcome, due to highly variable PRP quality among patients. Alternatively, allogeneic PRP from well-characterized donors cannot only generate more consistent and reliable therapeutic effect but also avoid harvesting large quantities of blood, an additional health burdens to patients. However, the use of allogeneic PRP for bone defect treatment is generally less investigated, especially for its immunogenicity in such application. Here, we meticulously investigated the immunogenicity of allogeneic PRP and evaluated its healing efficacy for critical-sized defect treatment. Allogeneic PRP contained 4.1-fold and 2.7- to 4.9-fold higher amount of platelets and growth factors than whole blood, respectively. The intramuscular injection of allogeneic PRP to rabbits did not trigger severe and chronic immunoresponse, evidenced by little change in muscular tissue microstructure and CD4⁺/CD8⁺ T lymphocyte subpopulation in peripheral blood. The implantation of allogeneic PRP/deproteinized bone matrix (DPB) constructs (PRP+DPB) successfully bridged 1.5-cm segmental radial defects in rabbits, achieving similar healing capacity as autologous MSC/DPB constructs (MSC+DPB), with greater bone formation (1.1-1.5×, p<0.05) and vascularization (1.3-1.6×, p<0.05) than DPB alone, shown by histomorphometric analysis, bone mineral density measurement, and radionuclide bone imaging. Furthermore, the implantation of both allogeneic PRP- and autologous MSC-mediated DPB constructs (PRP + MSC + DPB) resulted in the most robust bone regeneration (1.2-2.1×, p<0.05) and vascularization (1.3-2.0×, p<0.05) than others (PRP+DPB, MSC+DPB, or DPB alone). This study has demonstrated the promising use of allogeneic PRP for bone defect treatment with negligible immunogenicity, great healing efficacy, potentially more consistent quality, and no additional health burden to patients; additionally, the synergetic enhancing effect found between allogeneic PRP and autologous MSCs may shed a light on developing new therapeutic strategies for large bone defect treatment.

Cortical Bone Tissue Response of Injectable Octacalcium Phosphate-Hyaluronic Acid Complexes

We have previously shown that synthetic octacalcium phosphate (OCP) displays highly osteoconductive and biodegradable characteristics. However, OCP cannot be sintered without thermal decomposition due to the existence of water molecules in the structure. The acquisition of the moldability and the improvement of the handling property in this material are subjects for the clinical use. In the present study, we prepared OCP complex with hyaluronic acid (Hya) that could be used in the injectable form and further examined the bone tissue reaction to cortical bone by placing the complex directly on an 8-weeks-old ICR mouse calvaria in comparison with the placement of OCP granules only. The granule form of OCP (between 300 to 500 μm in diameter) was mixed with sodium hyaluronic acid with molecular weights 90 x 104. The complex revealed an injectable characteristic if it was utilized in a syringe. After polytetrafluoroethylen ring was mounted on mouse calvaria, the inner space of the ring was filled with the complex and left the complex as it is for 6 weeks. Histological examination using the decalcified specimens indicated that the OCP/Hya complex exhibited greater bone formation than OCP granules only group within the ring at 6 weeks. The results suggested that the OCP/Hya complex could be used as an injectable and osteoconductive bone substitute material in many clinical situations.

Effect of rhBMP-2 and VEGF in a vascularized bone allotransplant experimental model based on surgical neoangiogenesis

We have demonstrated survival of living allogeneic bone without long-term immunosuppression using short-term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study, bone morphogenic protein-2 (rhBMP-2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra-medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP-2 or rhBMP-2 + VEGF. FK-506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP-2 + VEGF) group than rhBMP-2 and control groups (p < 0.05). Osteoblast counts were also higher in the rhBMP-2 + VEGF group (p < 0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p = 0.059). Local administration of VEGF and rhBMP-2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts.

Efficient Bone Regeneration Induced by Bone Morphogenetic Protein-2 Released from Apatite-Coated Collagen Scaffolds

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. Clinically utilized BMP-2 uses a type-I collagen scaffold as a carrier. Here we hypothesized that an apatite coating on a type-I collagen scaffold would prolong the BMP-2 release period and enhance bone regeneration in calvarial defects in mice. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluid. BMP-2 release kinetics and bioactivity were evaluated by enzyme-linked immunosorbent assay and alkaline phosphatase activity measurement of cultured osteoblasts. Computed tomography and histomorphometry were performed eight weeks after various doses of BMP-2 were delivered to mouse calvarial defects using either non-modified or apatite-coated collagen scaffolds. Apatite-coated collagen scaffolds released 91.8 ± 11.5% of the loaded BMP-2 over 13 days in vitro, whereas non-modified collagen scaffolds released 98.3 ± 2.2% over the initial one day. The in vivo study showed that BMP-2 delivery with apatite-coated collagen scaffolds resulted in a significantly greater bone formation area and higher bone density than that with non-modified collagen scaffolds. This study suggests that simple apatite coating on collagen scaffolds can enhance the bone regeneration efficacy of BMP-2 released from collagen scaffolds.

Effect of increased strut porosity of calcium phosphate bone graft substitute biomaterials on osteoinduction

The effect of increasing strut porosity on the osteoinductivity of porous calcium phosphate (CaP) and silicate-substituted calcium phosphate (SiCaP) bone substitute materials was investigated in an ovine ectopic model. One to two millimeter-sized granules or block implants with strut porosities of 10, 20, or 30% were inserted into the left and right paraspinalis muscle. At 12 weeks, histological sections were prepared through the center of each implant and bone contact, bone area and implant area quantified. Backscattered scanning electron microscopy (bSEM) was used to visualize bone within small pores in the struts of the scaffolds. Increased bone formation was measured in the SiCaP with 30% strut porosity (5.482% ± 1.546%) when compared with the nonsilicate CaP with the same morphology (1.160% ± 0.502%, p = 0.02), indicating that silicate substitution may increase osteoinduction. Greater bone formation was seen in scaffolds with increased strut porosity. No bone growth was found in any of the SiCaP scaffold with 10% porosity. There was no significant difference between block and granule specimens. Scanning electron microscopy and EDX in combination with histology demonstrated bone formation within pores <5 μm in size. The use of silicate-substituted CaP material with increased strut porosity may further augment repair and regeneration in bony sites.

Calcium Sulfate with Stearic Acid as an Encouraging Carrier for Reindeer Bone Protein Extract.

Various bone proteins and growth factors in specific concentrations are required for bone formation. If the body cannot produce sufficient quantities of these factors, bone trauma can be healed with an implant that includes the required factors in a carrier. This study was designed to evaluate various calcium salt candidates that can be used as carrier with reindeer bone protein extract to induce ectopic bone formation in the muscle pouch model of mouse. The bone protein extract was either impregnated into the disc form of carrier or mixed with carrier powder before implantation. The radiographic analysis indicated increased bone formation in all of the active groups containing the bone protein extract compared to the controls within 21 days follow-up. The highest bone formation was seen in the group with calcium sulfate with stearic acid where new bone and calcified cartilage were clearly visible. The greatest bone formation occurred in the groups that had bone protein extract readily available. This indicates that the bone forming factors in sufficient concentrations are required at the early stage of bone formation. The calcium sulfate with stearic acid was the most suitable and effective carrier for reindeer bone protein extract.