Greater Bone Formation Research Articles

Effect of intermittent administration of adiponectin on bone regeneration following mandibular osteodistraction in rabbits

Adiponectin, a protein hormone produced and secreted exclusively by adipocytes, was reported to increase bone mass and stimulate angiogenesis. However, the effect of adiponectin on bone regeneration following distraction osteogenesis has not yet been reported. In this study, rapid unilateral mandibular osteodistraction (distraction rate = 2 mm/day) was applied in 24 New Zealand white rabbits. The animals were then randomly divided into groups A and B (n=12 for each group). At the 1st, 3rd, and 5th day of the distraction, 200 µl PBS and 200 µl PBS contained 2 µg rh-adiponectin was intermittently injected into the distraction gap in groups A and B, respectively. At 6 weeks after the end of distraction, the rabbits were sacrificed, and the distracted mandibles were harvested and processed for radiography, dual energy X-ray absorptiometry (DXA), micro-CT, histology, and three-point bend testing. Under a rapid distraction, immature bone formation was seen in the distracted callus from group A. However, much greater bone formation and higher bone mineral density (BMD) and bone mineral content (BMC) of the distracted callus were observed in group B. Such finding was confirmed by histological, micro-CT examinations, and mechanical strength test. The results of this study suggest that intermittent administration of adiponectin can promote bone regeneration following rapid mandibular osteodistraction in rabbits.

Increased training loads do not magnify cancellous bone gains with rodent jump resistance exercise

This study sought to elucidate the effects of a low- and high-load jump resistance exercise (RE) training protocol on cancellous bone of the proximal tibia metaphysis (PTM) and femoral neck (FN). Sprague-Dawley rats (male, 6 mo old) were randomly assigned to high-load RE (HRE; n = 16), low-load RE (LRE; n = 15), or sedentary cage control (CC; n = 11) groups. Animals in the HRE and LRE groups performed 15 sessions of jump RE during 5 wk of training. PTM cancellous volumetric bone mineral density (vBMD), assessed by in vivo peripheral quantitative computed tomography scans, significantly increased in both exercise groups (+9%; P < 0.001), resulting in part from 130% (HRE; P = 0.003) and 213% (LRE; P < 0.0001) greater bone formation (measured by standard histomorphometry) vs. CC. Additionally, mineralizing surface (%MS/BS) and mineral apposition rate were higher (50-90%) in HRE and LRE animals compared with controls. PTM bone microarchitecture was enhanced with LRE, resulting in greater trabecular thickness (P = 0.03) and bone volume fraction (BV/TV; P = 0.04) vs. CC. Resorption surface was reduced by nearly 50% in both exercise paradigms. Increased PTM bone mass in the LRE group translated into a 161% greater elastic modulus (P = 0.04) vs. CC. LRE and HRE increased FN vBMD (10%; P < 0.0001) and bone mineral content (∼ 20%; P < 0.0001) and resulted in significantly greater FN strength vs. CC. For the vast majority of variables, there was no difference in the cancellous bone response between the two exercise groups, although LRE resulted in significantly greater body mass accrual and bone formation response. These results suggest that jumping at minimal resistance provides a similar anabolic stimulus to cancellous bone as jumping at loads exceeding body mass.

Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/β-catenin canonical Wnt signaling

Diet and nutritional status are critical factors that influences bone development. In this report we demonstrate that a mixture of phenolic acids found in the serum of young rats fed blueberries (BB) significantly stimulated osteoblast differentiation, resulting in significantly increased bone mass. Greater bone formation in BB diet-fed animals was associated with increases in osteoblast progenitors and osteoblast differentiation and reduced osteoclastogenesis. Blockade of p38 phosphorylation eliminated effects of BB on activation of Wnt signaling in preosteoblasts. Knocking down β-catenin expression also blocked the ability of serum from BB diet-fed rats to stimulate osteoblast differentiation in vitro. Based on our in vivo and in vitro data, we propose that the underlying mechanisms of these powerful bone-promoting effects occur through β-catenin activation and the nuclear accumulation and transactivation of TCF/LEF gene transcription in bone and in osteoblasts. These results indicate stimulation of molecular events leading to osteoblast differentiation triggered by P38 MAP kinase (MAPK)/β-catenin canonical Wnt signaling results in significant increases in bone growth in young rats consuming BB-supplemented diets. Liquid chromatography/mass spectrometry (LC/MS) characterization of the serum after BB feeding revealed a mixture of simple phenolic acids that may provide a basis for developing a new treatment to increase peak bone mass and delay degenerative bone disorders such as osteoporosis.

Diseño de un programa de investigación sobre utilización de terapia celular y BMP en artrodesis lumbar

We report the spinal results of the use of bone morphogenetic protein (BMP-7) and pluripotent stem cells differentiated to the osteogenic lineage. The results of this randomized, controlled clinical trial show that BMP-7 achieved similar bone formation to the use of autograft. Equally, in another controlled prospective cohort, greater bone formation was found when BMP-7 was used with allograft compared with allograft alone. No adverse effects were found. Experimental studies of cell therapy show promising results. The current foundations of cell therapy are discussed.

Surface modification of PCL‐TCP scaffolds in rabbit calvaria defects: Evaluation of scaffold degradation profile, biomechanical properties and bone healing patterns

Traditionally, polycaprolactone (PCL) based scaffolds tend to degrade at a slow rate. Pretreatment of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds under alkaline conditions can be utilized to increase the degradation rate and improve mechanical properties. Three groups of PCL-TCP scaffolds with varying pretreatment exposures with sodium hydroxide (NaOH) were studied in a rabbit calvaria defect model and analyzed at 2, 4, 8, 12, and 24 weeks. (Group A: Untreated, Group B: 3 M NaOH/ 48 h and Group C: 3 M NaOH/96 h). Micro-CT analysis demonstrated that scaffolds with increased surface roughness (Groups B and C) showed a greater impact on the overall volume loss during the early healing period between 2 and 8 weeks as compared to the untreated group. In addition, greater bone formation was detected in NaOH treated scaffolds as compared to the untreated group throughout the experiment. Scaffolds with increased surface roughness generally reported higher push out test and compressive strength values from 4 to 8 weeks of early healing. Interestingly, the mechanical properties displayed a decline in values from 12 weeks onwards in the modified groups suggesting a favorable breakdown or weakening of PCL-TCP scaffolds tailored for replacement by new bone formation.

A biodegradable scaffold for the treatment of a diaphyseal bone defect of the tibia

The aim of this study was to compare angiogenesis and osteogenesis occurring within 8.0 mm diaphyseal defects created in canine tibiae treated using autograft or a biodegradable bone scaffold. All tibiae were reamed to 7.0 mm and fixed with a 6.5-mm statically locked intramedullary nail. Each of the 18 canines as allotted to one of three treatment groups: (1) left empty (N = 5), (2) treated with iliac crest autograft (N = 6), or (3) treated with a PLGA/calcium phosphate biodegradable scaffold (N = 7). Fluorescent markers were given at successive time periods: calcein green at 6 weeks, xylenol orange at 9 weeks, and tetracycline at 11 and 14 weeks. Animals were sacrificed at 15 weeks and their legs were perfused with a radio opaque compound. Samples were analyzed using Micro CT, bright-field microscopy and fluorescent microscopy. Scaffold samples were found to have significantly greater bone formation (p = 0.015) and blood vessel formation (p < 0.001) at their osteotomy sites than autograft samples. Bone formation rate in the periosteum was significantly greater in the autograft samples than the scaffold samples for all time periods. Bone formation at the osteotomy site was found to be significantly greater when associated with greater blood vessel formation (p = 0.026). The PLGA/calcium phosphate biodegradable scaffold we have employed supports angiogenesis within a segmental tibial defect that has adequate soft tissue coverage.

Orally Administered Lactoferrin Preserves Bone Mass and Microarchitecture in Ovariectomized Rats

Lactoferrin (LF) is reported to stimulate osteoblast proliferation and inhibit osteoclast activity in bone cell culture. However, the effect of oral LF on bone in osteoporosis needs to be explored. Three-month-old female Sprague-Dawley rats (n = 70) were assigned to the following groups: sham-operated, ovariectomized (OVX) untreated, OVX + bovine serum albumin (BSA; 85 mg/kg body weight), OVX + LF (0.85 mg/kg, 8.5 mg/kg, and 85 mg/kg body weight), and OVX + 17β-estradiol (E2; 10 μg/kg body weight). After 3 mo of treatment, E2 completely prevented the OVX-induced bone loss. OVX rats treated with LF were protected against the OVX-induced reduction of bone volume, trabecular number, and thickness, and the elevation of trabecular separation was prevented. LF also increased bone mineral density and increased the parameters of mechanical strength at 8.5- and 85-mg/kg doses. Greater bone formation and reduced bone resorption, as assessed by biochemical markers of bone remodeling, occurred in rats administered LF. LF at 8.5- and 85-mg/kg concentrations caused a significant decrease in serum calcium, but this reduction did not occur in rats fed 0.85 mg/kg LF. In addition, serum tumor necrosis factor-α and interleukin-6 production were suppressed and serum calcitonin was elevated significantly in LF-fed rats at all 3 doses. These findings indicated that oral LF not only preserved bone mass but also improved bone microarchitecture. The absorption of LF peptides and their effects on bone cells could to some extent account for the osteogenic function of oral LF.

Proportion of deproteinized bovine bone and autogenous bone affects bone formation in the treatment of calvarial defects in rabbits

This study assessed new bone formation generated using three different proportions of autogenous bone (AB) and deproteinized bovine bone (DBB). Thirty bicortical skull defects were prepared in 15 rabbits, divided into 3 groups: Group 1, critical size defect (CSD) versus AB as controls; Group 2, DBB versus a composite of AB and DBB using a proportion of 1:1; and Group 3, a composite of AB and DBB using a proportion of 1:2 versus a proportion of 1:4. After 8 weeks, radiographic evaluation was assessed using densitometry and new bone formation by histomorphometry. The mean optical density of the CSD (0.108 ± 0.238) and AB (0.352 ± 0.161) groups differed significantly from the DBB group (1.044 ± 0.093) and the groups using a proportion of 1:1 (0.905 ± 0.078), 1:2 (0.865 ± 0.294) and 1:4 (0.867 ± 0.304). Histomorphometry revealed a higher percentage of new bone in the AB group (30.223 ± 16.722) than in the groups using proportions of 1:2 (22.639 ± 5.659), 1:1 (20.929 ± 6.169), 1:4 (9.621 ± 2.400), DBB (14.441 ± 2.742) and CSD (10.645 ± 8.868), respectively. The 1:2 group had significantly higher bone content than the 1:4 group. The proportions of 1:1 and 1:2 resulted in greater bone formation than the proportion of 1:4, DBB and CSD.

Enhanced bone formation by marrow‐derived endothelial and osteogenic cell transplantation

Bone marrow-derived osteogenic cells can regenerate bone tissuesin vivo. The aim of the present study is to determine whether the cotransplantation of bone marrow-derived endothelial-like cells (BMECs) enhances bone regeneration by bone marrow-derived osteogenic cell (BMOC) transplantation in osseous defects. Canine bone marrow cells were differentiated separately into BMECs and BMOCs. Using apatite-coated poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds as cell delivery vehicles, BMOCs were transplanted with or without BMECs into critical-sized calvarial defects in immunodeficient mice. Histological analyses, microcomputed tomography, and soft X-ray were performed to assess mineralized bone formation at 8 weeks. Cotransplantation of BMECs and BMOCs resulted in greater bone formation than transplantation of BMOCs alone. There was a significant (p < 0.05) increase in bone formation area following cotransplantation (30.8% +/- 2.5%), compared with transplantation of BMOCs alone (15.3% +/- 1.9%). These results demonstrate that the cotransplantation of BMECs enhances bone regeneration mediated by BMOC transplantation in osseous defects.

In vitro dexamethasone pretreatment enhances bone formation of human mesenchymal stem cells in vivo

Bone grafting is the current standard of care for treatment of fracture nonunions, while alternative strategies such as bone marrow-derived mesenchymal stem cells are also used. MSCs can be induced towards the osteogenic lineage by in vitro treatment with dexamethasone (dex). This study aimed to determine the optimal duration of dex treatment for osteogenic differentiation of MSCs in vitro and evaluate the effect of this dex pretreatment on bone formation in vivo. To determine the optimal dex treatment, MSCs were cultivated in osteogenic medium for 5 weeks with a varying dex withdrawal schedule, such that MSCs were exposed to dex for either 0, 1, 2, 3, 4, or 5 weeks. During this period, alkaline phosphatase, calcium, and DNA assays, as well as von Kossa staining and morphological observations were performed. One and 2 week dex-treated groups returned to control levels rapidly, whereas 3 and 4 week groups retained higher levels of differentiation markers, with the 4 week group being the highest. Based on these in vitro results, MSCs (with and without dex) and control fibroblasts were seeded into ceramic cubes, cultured for 4 weeks, and implanted into SCID mice, and harvested 6 weeks postimplantation for histologic evaluation. There was no bone formation in fibroblast-seeded controls, little bone formation in control (CS 1), and extensive bone formation (CS 3-4) in dex-treated MSCs. These results indicate that pretreatment of MSCs with dex results in greater bone formation than in untreated controls.

A biodegradable scaffold for the treatment of a diaphyseal bone defect of the tibia

The aim of this study was to compare angiogenesis and osteogenesis occurring within 8.0 mm diaphyseal defects created in canine tibiae treated using autograft or a biodegradable bone scaffold. All tibiae were reamed to 7.0 mm and fixed with a 6.5-mm statically locked intramedullary nail. Each of the 18 canines as allotted to one of three treatment groups: (1) left empty (N = 5), (2) treated with iliac crest autograft (N = 6), or (3) treated with a PLGA/calcium phosphate biodegradable scaffold (N = 7). Fluorescent markers were given at successive time periods: calcein green at 6 weeks, xylenol orange at 9 weeks, and tetracycline at 11 and 14 weeks. Animals were sacrificed at 15 weeks and their legs were perfused with a radio opaque compound. Samples were analyzed using Micro CT, bright-field microscopy and fluorescent microscopy. Scaffold samples were found to have significantly greater bone formation (p = 0.015) and blood vessel formation (p < 0.001) at their osteotomy sites than autograft samples. Bone formation rate in the periosteum was significantly greater in the autograft samples than the scaffold samples for all time periods. Bone formation at the osteotomy site was found to be significantly greater when associated with greater blood vessel formation (p = 0.026). The PLGA/calcium phosphate biodegradable scaffold we have employed supports angiogenesis within a segmental tibial defect that has adequate soft tissue coverage.

Ti Alloy Surface Modifications and Coatings: An Update

Since 1952 when Branemark first reported osseointegration of titanium (Ti) with bone, many academic and industrial research activities have endeavored to improve the efficacy of Ti or Ti alloy (Ti6Al4V) by modifying the chemistry, topography and design of the implant surface. Strong bonding between implant and host tissue minimize the micromovements that promote fibrous tissue formation at the implant interface that may lead to implant failure. Surface design include lateral holes perpendicular to the implant axis, grooves, variations of spacings between ridges, etc. Physico-mechanical means of surface modification is by grit-blasting with various abrasives (alumina, silica, apatitic abrasive), laser ablation, spark discharge, etc. Chemical modifications include: acid etching, treatment with alkali, treatment with fluoride, coating with titanium or with calcium phosphate (by plasma spray, electromagnetic sputtering, electrochemical deposition). A review of studies on Ti or Ti alloy implants with different surfaces showed the following methods to enhance osseointegration and greater bone formation: (1) grit-blasting with apatitic abrasive; (2) acid-etching with mixed acids; (3) adjusting plasma-spray parameter to get a higher HA/ACP ratio in the coating; (4) employing electrochemical deposition (with pulse modulation) or precipitation to obtain thin coating with homogeneous composition; and/or (5) Ftreatment.

Optimal Low-Energy Laser Irradiation Causes Temporal G2/M Arrest on Rat Calvarial Osteoblasts

Low-energy laser irradiation (LELI) accelerates wound healing and is thought to accelerate bone formation. However, the mechanism of laser healing is not clear. To clarify the biological mechanism of LELI healing, we investigated the effects of LELI on rat osteoblasts in vitro. Osteoblastic cells from 3-day-old Wistar rat calvaria were irradiated using a low-energy gallium-aluminum-arsenide (Ga-Al-As) diode laser. Bone formation, osteoblast differentiation, and cell proliferation were evaluated by von Kossa staining, reverse-transcription polymerase chain reaction, alkaline phosphatase (ALP) staining, 5-bromo-2'-deoxyuridine (BrdU) uptake, and fluorescence-activated cell sorter (FACS) analysis. At 21 days after LELI, the greatest bone formation was observed with irradiation energy of 3.75 J/cm2 and the first week after seeding. LELI (3.75 J/cm2) induced an increased number of cells at day 3. LELI-stimulated differentiation in osteoblastic cells was demonstrated by the increases of Runx2 expression and ALP-positive colonies. By contrast, at 1 day after laser irradiation, the number of cells in the irradiation group was significantly lower than that in the control group. BrdU uptake indicated lower proliferation 12 and 24 hours after irradiation compared with the control. Furthermore, FACS data demonstrated a higher proportion of cells in the G2/M phase of the cell cycle 12 hours after irradiation compared with the control. G2/M arrest was confirmed by the appearance of G2/M arrest marker 14-3-3-sigma or phospho-p53. These results demonstrate that LELI induces not only acceleration of bone formation but also initial G2/M arrest, which may cause wound healing like tissue repair.

Teriparatide Increases Bone Formation in Modeling and Remodeling Osteons and Enhances IGF-II Immunoreactivity in Postmenopausal Women With Osteoporosis

Transiliac bone biopsies were obtained from 55 women treated with teriparatide or placebo for 12-24 months. We report direct evidence that modeling bone formation at quiescent surfaces was present only in teriparatide-treated patients and bone formation at remodeling sites was higher with teriparatide than placebo. Recombinant teriparatide [human PTH(1-34)], a bone formation agent for the treatment of osteoporosis when given once daily subcutaneously, increases biochemical markers of bone turnover and activation frequency in histomorphometry studies. We studied the mechanisms underlying this bone-forming action of teriparatide at the basic multicellular unit by the appearance of cement lines, a method used to directly classify surfaces as modeling or remodeling osteons, and by the immunolocalization of IGF-I and IGF-II. Transiliac bone biopsies were obtained from 55 postmenopausal women treated with teriparatide 20 or 40 microg or placebo for 12-24 months (median, 19.8 months) in the Fracture Prevention Trial. A dose-dependent relationship was observed in modeling and mixed remodeling/modeling trabecular hemiosteons. Trabecular and endosteal hemiosteon mean wall thicknesses were significantly higher in both teriparatide groups than in placebo. There was a dose-dependent relationship in IGF-II immunoreactive staining at all bone envelopes studied. The greater local IGF-II presence after treatment with teriparatide may play a key role in stimulating bone formation. Direct evidence is presented that 12-24 months of teriparatide treatment induced modeling bone formation at quiescent surfaces and resulted in greater bone formation at remodeling sites, relative to placebo.

Overexpression of Runx2 directed by the matrix metalloproteinase‐13 promoter containing the AP‐1 and Runx/RD/Cbfa sites alters bone remodeling in vivo

The activator protein-1 (AP-1) and runt domain binding (Runx/RD/Cbfa) sites and their respective binding proteins, c-Fos/c-Jun and Runx2 (Cbfa1), regulate the rat matrix metalloproteinase-13 (MMP-13) promoter in both parathyroid hormone (PTH)-treated and differentiating osteoblastic cells in culture. To determine the importance of these regulatory sites in the expression of MMP-13 in vivo, transgenic mice containing either wild-type (-456 or -148) or AP-1 and Runx/RD/Cbfa sites mutated (-148A3R3) MMP-13 promoters fused with the E. coli lacZ reporter were generated. The wild-type transgenic lines expressed higher levels of bacterial beta-galactosidase in bone, teeth, and skin compared to the mutant and non-transgenic lines. Next, we investigated if overexpression of Runx2 directed by the MMP-13 promoter regulated expression of bone specific genes in vivo, and whether this causes morphological changes in these animals. Real time RT-PCR experiments identified increased mRNA expression of bone forming genes and decreased MMP-13 in the tibiae of transgenic mice (14 days and 6 weeks old). Histomorphometric analyses of the proximal tibiae showed increased bone mineralization surface, mineral apposition rate, and bone formation rate in the transgenic mice which appears to be due to decreased osteoclast number. Since MMP-13 is likely to play a role in recruiting osteoclasts to the bone surface, decreased expression of MMP-13 may cause reduced osteoclast-mediated bone resorption, resulting in greater bone formation in transgenic mice. In summary, we show here that the 148 bp upstream of the MMP-13 transcriptional start site is sufficient and necessary for gene expression in bone, teeth, and skin in vivo and the AP-1 and Runx/RD/Cbfa sites are likely to regulate this. Overexpression of Runx2 by these regulatory elements appears to alter the balance between the bone formation-bone resorption processes in vivo.

Endothelial cell modulation of bone marrow stromal cell osteogenic potential

In the context of bone development and regeneration, the intimate association of the vascular endothelium with osteogenic cells suggests that endothelial cells (ECs) may directly regulate the differentiation of osteoprogenitor cells. To investigate this question, bone marrow stromal cells (BMSCs) were cultured: in the presence of EC-conditioned medium, on EC extracellular matrix, and in EC cocultures with and without cell contact. RNA and protein were isolated from ECs and analyzed by reverse transcriptase-polymerase chain reaction and Western blotting, respectively, for expression of bone morphogenetic protein 2 (BMP-2). In animal studies, BMSCs and ECs were cotransplanted into severe combined immunodeficient mice on biodegradable polymer matrices, and histomorphometric analysis was performed to determine the extent of new bone and blood vessel formation. ECs significantly increased BMSC osteogenic differentiation in vitro only when cultured in direct contact. ECs expressed BMP-2, and experiments employing interfering RNA inhibition confirmed its production as contributing to the increased BMSC osteogenic differentiation. In vivo, cotransplantation of ECs with BMSCs resulted in greater bone formation than did transplantation of BMSCs alone. These data suggest that ECs function not only to form the microvasculature that delivers nutrients to developing bone but also to modulate the differentiation of osteoprogenitor cells in vitro and in vivo.

Long-term results of segmental prosthesis fixation by extracortical bone-bridging and ingrowth.

The technique of extracortical bone-bridging and ingrowth fixation with a porous coating over the shoulder region of the implant and augmentation by autogenous bone-grafting was introduced to improve the longevity of implant fixation. The potential advantages of this technique are that new-bone formation across the bone-prosthesis junction may share stress and may prevent osteolysis by sealing off this critical region against the infiltration of wear particles. The objectives of this study were to examine the prevalence of stem-loosening with use of the extracortical bone-bridging and ingrowth technique, the amount of bone formation over the porous-coated region of this prosthesis, and the characteristics of bone formation over the porous-coated region and adjacent bone. Forty-three patients who had prosthetic reconstruction with the extracortical bone-bridging and ingrowth technique from 1976 to 1990 were included in this retrospective study. The mean length of follow-up was 9.7 years (range, two to twenty-one years). All but one patient were managed with autogenous bone graft; five, with allograft and autograft; and one, with allograft only. Extracortical bone formation was measured over a 2-cm length of the porous-coated region of the prosthesis in four zones (the medial and lateral aspects on anteroposterior radiographs and the anterior and posterior aspects on lateral radiographs) and was reported as the percentage of the total length (8 cm) covered by extracortical bone with a thickness of >1 mm. The Spearman rank coefficient was used to assess the correlation between pairs of continuous variables. The final average percentage of the porous-coated region that was covered by extracortical bone formation was 76% +/- 34% for all patients and anatomical sites of reconstruction. Use of bone cement was associated with less bone formation (p = 0.04), and this value remained lower at the final measurement (p = 0.06). One stem had aseptic loosening, but no sign of osteolysis was found. The radiographic appearance of the bone formation had stabilized at two years of follow-up. All patients with allograft augmentation had greater bone formation. The amount of extracortical bone formation did not differ in relation to the type of porous coating, anatomical sites, pathological disorder, sex or age of the patient, or length of reconstruction. As shown by the low prevalence of stem-loosening (two of fifty-six stems or one of forty-three patients), the use of the extracortical bone-bridging and ingrowth fixation technique is associated with improved stem fixation in segmental bone-replacement prostheses applied for limb salvage. In the demanding biomechanical environment and with the risk of stress and particle-related bone resorption, the extracortical bone-bridging and ingrowth fixation is an attractive method to provide long-lasting implant fixation.

Subperiosteal tissue expansion: an experimental study

Periosteal flaps have been used to cover bone defects. There are no sufficient data on whether expanded periosteum can be used to repair bone defects after subperiosteal tissue expansion. In this experimental study, 14 male dogs, which were 2 years-old and weighed 14.5–16.0 kg, were used to investigate the repair of bony defects with expanded periosteum. Rectangular tissue expanders, measuring 50 mm in width, 70 mm in length, and 20 mm in height (volume: 20 ml) were used. The dogs were divided into two groups each of seven dogs: one control group and one experimental group. In each dog, a pocket, 50×70 mm in size, was prepared in parietal area, and a periosteal area, 30×40 mm in size, was marked out. A rectangular tissue expander was inserted into the pocket. In the control group, the tissue expanders were not inflated. On the seventh postoperative day, the tissue expander inflation with normal saline (5 ml per 2 days) was started in the experimental group. After 15 days, the previously marked out periosteal area was measured. A specimen was taken from this periosteal tissue for histopathological evaluation. A bone defect, 20×20 mm in size, was created beneath expanded periosteal tissue, and this defect was covered with the expanded periosteal tissue. After 15 days, a histopathological evaluation was carried out. Statistical analysis was carried out using the Mann–Whitney U-test. The area of periosteum expanded was more in the experimental group than that of the control group (p>0.05). After removal of the tissue expander, both in the control and subperiosteal tissue expansion group, osteoblastic lining of the expanded periosteum, increased vascularity, and granulation tissue was noted. Following 15 days, woven and lamellated bone tissue was formed both in the control and subperiosteal expansion group. In the subperiosteal expansion group, there was greater bone formation, the expanded periosteum was thickened, and thickened lamellated bone was covered by active osteoblasts. It is concluded that subperiosteal tissue expansion may be worthwhile to provide large periosteal flaps for the repair of large bone defects, by increasing osteogenic capacity.

Dietary Essential Amino Acid Supplements Increase Bone Strength by Influencing Bone Mass and Bone Microarchitecture in Ovariectomized Adult Rats Fed an Isocaloric Low-Protein Diet

This study was designed to investigate whether the administration of dietary essential amino acid supplements in adult rats made osteoporotic by estrogen deficiency and reduced protein intake could reverse the deleterious effects caused by these maneuvers. This animal model was selected to mimic the situation observed in elderly women in whom estrogen deficiency and/or low-protein intake (but also calcium and vitamin D deficiency) are known to contribute to the pathogenesis of osteoporosis. Six-month-old rats were ovariectomized (OVX) and fed an isocaloric 2.5% casein diet for 10 weeks or sham-operated (SHAM) and fed an isocaloric 15% casein diet. The animals fed the 2.5% casein diet were given isocaloric supplements of essential amino acids in similar relative proportion to that of casein at doses of 2.5% or 5% of total diet for an additional 16 weeks. Vertebrae, femur, and tibia bone mineral density (BMD); ultimate strength; and microtomographic histomorphometry were evaluated before and after dietary essential amino acid supplements. Essential amino acid supplements increased vertebrae, femur, and tibia bone strength in OVX rats fed a low-protein diet. The mechanical changes induced by this dietary isocaloric supplement were associated with the prevention of a further BMD decrease or even with some increases and changes in microarchitecture such as from a rod to a plate trabecular spacial configuration and increased cortical thickness. Higher insulin-like growth factor (IGF) I levels, as well as greater bone formation and reduced bone resorption as assessed by biochemical markers of bone remodeling, were found in rats receiving essential amino acid supplements. In conclusion, dietary essential amino acid supplements increased bone strength through modifications of BMD, trabecular architecture, and cortical thickness possibly by an IGF-I-mediated process.

In vivo and in vitro biodegradation of oxychitin–chitosan and oxypullulan–chitosan complexes

Oxychitin–chitosan complexes prepared from crustacean chitin and oxypullulan–chitosan complexes prepared from three different preparations of Aureobasidium pullulans pullulan were contacted with solutions of egg white lysozyme, Carica papaya papain, wheat germ lipase, Clostridium histolyticum collagenase, porcine pancreas α-amylase, barley malt α-amylase, and sweet potato α-amylase at nearly neutral pH values and 25 and 37°C, for at least three days. The reducing capacity of the solutions in contact with the complex, due to oligomer release, was measured with the aid of ferricyanide and expressed as net absorbance vs time. The oxychitin–chitosan complex was degraded by lysozyme, lipase and papain. The other enzymes were ineffective. Histological evidence indicated that the oxychitin–chitosan complex tested as a bone prosthesis coating in an animal model, was biochemically active and biodegradable, therefore capable to promote osteoconduction and greater bone formation at the bone-prosthesis interface, with no adverse effect on mineralization. Pullulans of different origins were susceptible to enzymatic hydrolysis, particularly so with animal amylase, nevertheless the chitosan complexes obtained from the corresponding oxypullulans were not degraded over the three-day observation period.