Locally delivered rhBMP-2 enhances bone ingrowth and gap healing in a canine model.

The purpose of the present study was to determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) enhances bone ingrowth into porous-coated implants and gap healing around the implants. In the presence of a 3-mm gap between the implant and host bone, porous-coated implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs. In three treatment groups, the test implant was treated with HA/TCP and rhBMP-2 in buffer at a dose of 100 microg/implant (n=5), 400 microg/implant (n=6), or 800 microg/implant (n=5) and placed in the left humerus. In these same animals, an internal control implant was treated only with HA/TCP and buffer and placed in the right humerus. These groups were compared with a previously reported external control group of seven animals in which no growth factor was delivered [J. Orthop. Res. 19 (2001) 85]. The BMP treated implants in the two lower dose groups had significantly more bone ingrowth than the external controls with the greatest effect in the 100 g/implant group (a 3.5-fold increase over the external control, p=0.008). All three dose groups had significantly more bone formation in the 3-mm gap surrounding the BMP treated implants than the external controls with the greatest effect in the 800 microg group (2.9-fold increase, p<0.001). Thus, application of rhBMP-2 to a porous-coated implant stimulated local bone ingrowth and gap healing. The enhancement of bone formation within the implant (bone ingrowth) was inversely related to dose.     

Combination of local and systemic parathyroid hormone enhances bone regeneration

Parathyroid hormone is one of the most promising therapeutic agents for osteoporosis, but its use to facilitate bone regeneration in osseous defects is less clear. The purpose of the current study was to determine the effects of combining systematic parathyroid hormone and a local parathyroid hormone gene therapy in a critical-sized osteotomy model. Rats received bilateral femoral osteotomies followed by implantation of a gene-activated matrix encoding parathyroid hormone (1-34) on one side and a control gene-activated matrix on te opposite side. Systematic parathyroid hormone (1-34) or vehicle was injected daily and rats were sacrificed 6 weeks later. Systematic parathyroid hormone increased bone mineral density and bone mineral content measured by dual-energy xray absorptiometry analysis of tibias and vertebrae, and increased serum osteocalcin levels during healing of osteotomies. Furthermore, comparing osteotomy sites that received the same gene-activated matrices as vehicle-injected rats, parathyroid hormone-injected rats showed trends of greater bone areas via histomorphometric and microradiographic analyses and higher osteocalcin messenger ribonucleic acid expression via Northern blot analyses. The combination of systemic and local parathyroid hormone led to higher bone mineral density, bone mineral content, and bone area, a trend for greater radiographic-detected bone area and higher expression of osteocalcin in osteotomy sites when compared with the individual treatment or control groups. Local parathyroid hormone gene therapy enhanced the anabolic effect of systemic parathyroid hormone during osteotomy healing. This study supports the concept of a combined local and systemic approach for enhancing the repair of a fracture at risk for nonunion.     

Local application of rhTGF-beta2 enhances peri-implant bone volume and bone-implant contact in a rat model

Orthopedic and dental implant fixation depends upon bone regeneration. Growth factors such as transforming growth factor-beta (TGF-beta) have been shown to enhance bone repair and strengthen the mechanical connection between implant and host skeleton in canine models. To provide a platform for studying molecular mechanisms of growth factor stimulated bone regeneration and implant fixation, the present study examined peri-implant bone volume as a response to TGF-beta treatment in a rodent model. The rat femoral ablation model in which an implant is placed in the medullary cavity of the femur was used to examine the dose response to TGF-beta2 applied to the implant (0, 0.1, 1.0, or 10 microg). The study included a total of 40 rats (10 per dose) examined at 28 days. Peri-implant bone volume and bone-implant contact were assessed through microcomputed tomography and implant fixation strength was determined by a mechanical pullout test. Treatment of the implant with 10 microg TGF-beta2 led to a 2-fold increase in bone volume (P<0.001) and a 1.5-fold increase in bone-implant contact (P<0.01) with a trend of increasing fixation strength (non-significant increase of 1.4-fold). TGF-beta2 treatment with 10 microg led to uniform peri-implant bone volume and bone-implant contact along the length of the implant, whereas the other groups had less bone at the mid-point compared to the proximal and distal aspects of the implant. About 50% of the variance in implant fixation strength was explained by a regression model involving both bone-implant contact and peri-implant bone volume.     

Osteogenesis by recombinant human bone morphogenetic protein-2 at skeletal sites

Osteogenesis was evaluated in the mandibular bone by combinations of various dosages of recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite (four groups: Group I, 2 micrograms recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Group II, 10 micrograms recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Group III, 50 micrograms recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Control Group, only atelopeptide Type I collagen and porous hydroxyapatite). The prepared materials were implanted in the mandibular bone hole (7 mm in diameter, 2 mm deep). Three weeks later, the alkaline phosphatase activity in the implanted region was determined, and the histologic features of the excised tissue were examined. There were significant differences in histologic and biochemical findings among the four groups. In the recombinant human bone morphogenetic protein-2 implanted groups, osteogenesis increased with the dosage of recombinant human bone morphogenetic protein-2, as assessed by alkaline phosphatase activity and histologic findings. The results suggest that atelopeptide Type I collagen is an effective carrier for recombinant human bone morphogenetic protein-2 and that porous hydroxyapatite would be advantageous for clinical application as a material to maintain its original form after implantation.     

Controlled release of platelet growth factors enhances bone regeneration at rabbit calvaria.

OBJECTIVE: Platelet-rich plasma (PRP) has been clinically employed to promote bone regeneration. However, few studies have investigated the enhancement of biological function of platelet growth factors after integration of PRP into biomaterials. In this study, the feasibility of gelatin hydrogels for controlled release of platelet growth factors and the consequent enhancement of PRP-induced bone regeneration were evaluated in rabbit calvarial defect. STUDY DESIGN: Gelatin hydrogels incorporating PRP, PRP-activated thrombin, or an empty gelatin hydrogel were applied to the defect, or the defect was left untreated. Bone regeneration was evaluated by microfocus computed tomography, peripheral quantitative computed tomography, and histological examinations. RESULTS: Successful bone regeneration was observed at the bone defect applied with the gelatin hydrogel incorporating PRP, which is in marked contrast to other groups. CONCLUSION: The gelatin hydrogel is a promising material capable of controlled release of platelet growth factors to enhance bone regeneration.     

Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration.

Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions. INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation. MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT. RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition. CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics.     

North American male reference population for speed of sound in bone at multiple skeletal sites.

Alternatives to dual-energy X-ray absorptiometry (DXA) have been sought to increase access to low-cost osteoporosis risk assessment. Early quantitative ultrasound (QUS) systems measured speed of sound (SOS) and broadband ultrasound attenuation (BUA) at the calcaneus, and these were demonstrated to be good predictors of hip fracture risk. Recent studies have demonstrated the usefulness of other peripheral sites to assess bone status. The Sunlight Omnisense (Sunlight Medical, Rehovot, Israel) is a portable, inexpensive QUS device capable of multiple-site SOS measurement. To provide a robust male reference database, 588 healthy Caucasian males aged 20-90 yr were recruited from 6 centers across North America. SOS measurements were taken at the distal 1/3 radius, proximal third phalanx, midshaft tibia, and fifth metatarsal. A female reference database has previously been collected at North American sites. The results indicate that SOS in males exhibits an age-related decline beginning in the fifth decade at the radius, phalanx, and metatarsal, whereas the tibial SOS remains nearly constant until the ninth decade. Although females reach a higher-peak SOS than males at most sites, SOS is higher in males at all sites after the sixth decade, as a result of a more gradual decline in SOS. Longitudinal monitoring of healthy men should be performed to confirm these cross-sectional results.     

rhBMP-2诱导界面骨长入中空金属植入物的初步实验研究

目的：探讨界面骨质经孔洞长入中空金属植入物及以rhBMP-2/载体复合物诱导增效的可能性。方法：以兔胫骨干骺端植入集骨器（bone harvest chamber,BHC)为动物模型，在其中分别加载rhBMP-2和rhBMP-2/透明质酸钠凝胶（Healon GV)复合物10μl（均含rhBMP-2 1mg)，并以单纯置入等量Healon GV和注射用水作为对照。2,4,6周取出BHC中组织，行组织学观察并行MMA包埋不脱钙骨切片，计算机图像分析测算小梁骨总体积密度（trabecular bone volume,TBV)进行骨形态计量学分析，组间比较成骨量。结果：rhBMP-2/Healon GV复合物组和rhBMP-2组于植入2周后BHC内均已有交织骨形成，前者骨质较密集；4周时为成熟骨小梁间以髓样组织填充；6周时骨质更为密集。而Healon GV组和注射用水组4周的表现与前两组2周时接近，6周时才形成成熟的骨小梁和髓样组织。结论：界面骨质可通过也洞长入中空金属植入物的空腔内并形成成熟的骨质，rhBMP-2可促进骨长入效应及成骨质量，rhBMP-2载体Healon GV不影响且可增强骨诱导作用。     

Ischemic preconditioning of skeletal muscle mitigates remote injury and mortality

BACKGROUND: Ischemic preconditioning (IPC) mitigates ischemia-reperfusion (I/R) injury in experimental models. However, the clinical significance of this protection has been unclear and a mortality reduction has not been previously reported in noncardiac models. This study examined the local and remote protection afforded by skeletal muscle IPC and sought to determine the significance of this protection on mortality. METHODS: Mice subjected to 2 h hindlimb ischemia/24 h reperfusion (standard I/R injury) were compared with those undergoing a regimen of two 20-min cycles of IPC followed by standard I/R injury. Local injury was assessed via gastrocnemius histology, and remote injury was evaluated via intestinal histology and pulmonary neutrophil infiltration (n = 7). Mortality was compared in parallel groups for 1 week (n = 6). Groups were analyzed using an unpaired Student's t-test for gastrocnemius and pulmonary injury, and a Mann-Whitney rank sum test for intestinal injury. Mortality differences were interpreted through a hazard ratio. RESULTS: Significant protection was observed in preconditioned animals. There was a 35% local injury reduction in skeletal muscle (71.2% versus 46.0%, P < 0.01), a 50% reduction in remote intestinal injury (2.3 versus 1.1, P < 0.01), and a 43% reduction in remote pulmonary injury (14.9 versus 8.5, P < 0.01) compared with standard injury controls. Preconditioned animals were also significantly protected from mortality, demonstrating a 66.7% survival at 1 wk compared with 0% survival after standard injury alone (hazard ratio 0.20, 95% CI: 0.02-0.59). CONCLUSIONS: We have developed a murine model of IPC that demonstrates local and remote protection against I/R injury, and exhibits significant mortality reduction. This model demonstrates the powerful effect of IPC on local and remote tissues and will facilitate further study of potential mechanisms and therapies.     

南京地区成年女性衰老过程中各部位骨密度的变化

目的 探讨南京地区成年女性骨质疏松的患病情况,及衰老过程中各部位骨密度的变化。方法 对南京地区成年女性,20-29岁,共计388人,均排除继发性骨质疏松症,运用双能X线骨密度仪,对所有患者进行腰椎及髋部骨密度测定。结果 1.南京地区成年女性,骨量正常者占33.5%,骨量低下者占48.5%,骨质疏松者占18%;其中绝经前成年女性骨量正常者占52.7%,骨量低下者占47.3%,无骨质疏松患者;绝经后女性骨量正常者占12%,骨量低下者占49.7%,骨质疏松患者占38.3%;2. 20~39岁年轻女性,随年龄增加,各部位骨密度均无明显下降;但自40岁开始至49岁,股骨颈骨密度首先开始下降,而此时腰椎及总髋部骨密度无明显变化;到50岁开始,各部位骨密度均明显下降;其中50~69岁时,腰椎骨密度T值与股骨颈骨密度T值之间的差异消失,但均低于总髋部骨密度T值。结论 1.南京地区年轻女性骨量低下者患病率较高,随年龄增加,绝经后女性骨量低下者进一步增多,且骨质疏松患病率高达38.3%;2.在女性50岁前,股骨颈骨密度下降最早,诊断骨质疏松最敏感,而50岁后,腰椎骨密度与股骨颈骨密度均明显下降,诊断骨质疏松同样敏感。     

Remodeling and ingrowth of bone at two years in a canine cementless total hip-arthroplasty model.

Remodeling and ingrowth of bone in association with the use of uncemented femoral components were examined at two years in a canine total hip-arthroplasty model. Twenty-two dogs received a unilateral uncemented femoral stem that was made of Ti6A14V and was covered with one of three types of titanium porous coating: fiber-metal, beads, or plasma flame-spray. The amount and distribution of ingrowth of bone differed somewhat among the groups at two years, but the patterns of remodeling of bone in the medullary canal and cortex were similar. In general, about 15 to 18 per cent of the cortical bone was lost adjacent to the levels of the stem that were covered with the porous coating. Most of the loss of cortical bone was due to subperiosteal resorption proximally and endosteal resorption at the middle and distal levels of the stem. Increased cortical porosity accounted for only a small fraction of the loss of cortical bone. The amount of medullary bone increased proximally and distally, so that the loss of total bone mass was significantly only at the mid-part of the stem. The amount of loss of cortical bone was similar to that observed in a previous six-month study, suggesting that a steady state was achieved in the present model.     

A novel, non-prostanoid EP2 receptor-selective prostaglandin E2 agonist stimulates local bone formation and enhances fracture healing.

CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective PGE2 agonist, stimulates local bone formation and enhances fracture healing in rat models. INTRODUCTION: There is a significant medical need for agents that can stimulate local bone formation and enhance fracture healing. We tested the effects of CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective prostaglandin E2 (PGE2) agonist, in stimulating local bone formation and enhancing fracture healing in rat models. MATERIALS AND METHODS: In the first model, a single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis of 6-week-old male rats was given on day 1, and the local bone anabolic effect was determined on day 7. We then tested the effects of this compound in inducing bone formation on rat periosteum of the femur. A single dose of 0.3 mg of CP-533,536 incorporated in a poly-(D,L-lactide-co-glycolide) (PLGH) matrix was injected onto the periosteum of the femur in 3-week-old male rats, and local bone formation was determined on day 14. Finally, the ability of CP-533,536 in PLGH matrix in enhancing fracture healing was tested using the rat femoral fracture model. CP-533,536 in PLGH matrix at doses of 0.05, 0.5, or 5 mg was delivered to the local fracture site on the same day of fracture, and its efficacy was evaluated on day 21. RESULTS AND CONCLUSIONS: A single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis dose-dependently stimulated local lamellar bone formation on trabecular, endocortical, and periosteal surfaces, and thus increased bone mineral content and bone strength at the injected site. Similarly, a single injection of 0.3 mg of CP-533,536 incorporated in PLGH matrix onto the periosteum of the femur induced significantly local bone formation. In the rat femoral fracture model, CP-533,536 in PLGH matrix at doses of 0.05, 0.5, and 5 mg dose-dependently increased callus size, density, and strength compared with PLGH matrix alone. These results show that CP-533,536 stimulates new bone formation on trabecular, endocortical, and periosteal surfaces and enhances fracture healing. These data reveal that EP2 receptor-selective agonists provide therapeutic potential for local bone augmentation, bone repair, and bone healing in humans.     

Relationship between bone mass and rates of bone change at appendicular measurement sites.

The rate of bone change among postmenopausal women may vary depending upon the initial bone mass. Examining this possibility is difficult, however, because of a negative statistical bias that occurs when change is regressed against the initial value of the same variable. In this article, four statistical methods were applied to measure the association between bone mass and the rate of bone change. The study population was Japanese-American women, who were monitored for approximately 5 years. Bone changes were determined for the calcaneus and the distal and proximal radius. The results were consistent across the bone sites but differed between statistical methods. Three of the four methods indicated that the women with the greater bone mass had the greater loss rates. The fourth method did not support this association. Possible reasons for the discordant results are discussed. Using the "best" estimate of the relationship, a gradual convergence of bone mass was projected over time toward the population mean. The convergence occurred because women with higher bone mass had a somewhat faster loss rate than women with lower bone mass. Overall, however, the variation in bone mass between individuals was large compared to the rate of convergence.     

Locally applied nerve growth factor enhances bone consolidation in a rabbit model of mandibular distraction osteogenesis.

Distraction osteogenesis is widely used in treating deformities, defects, and fractures of both long bones and craniofacial bones. Demands for acceleration of bone consolidation are increased in distraction osteogenesis. Nerve growth factor (NGF) can enhance innervation and bone regeneration in a fracture model and stimulate differentiation of osteoblastic cells. In this study, we tested the ability of locally applied NGF to enhance bone regeneration in a rabbit model of mandibular distraction osteogenesis. Twenty rabbits underwent bilateral distraction osteogenesis with a rate of 0.5 mm per 12 h. Two times 0.04 mg human NGFbeta (hNGFbeta) in buffer was injected into the callus after distraction. The contralateral side received placebo injections. Rabbits were euthanized at consolidation times of 14 and 28 days. Specimens were subjected to radiography, callus dimensions measurement, mechanical testing, and bone histological and histomorphometric analysis. The maximum load, bone volume/total volume, mineral apposition rate of the 1st to 11th day, and mineralized bone percentage were significantly higher in the hNGFbeta side at 14 and 28 days (p<0.05). The data indicate that locally applied hNGFbeta can accelerate callus maturation and may be an option to shorten the consolidation period in distraction osteogenesis.     

Administration of tauroursodeoxycholic acid enhances osteogenic differentiation of bone marrow-derived mesenchymal stem cells and bone regeneration

It is known that osteogenic differentiation of mesenchymal stem cells (MSCs) can be promoted by suppression of adipogenesis of MSCs. We have recently found that the chemical chaperone tauroursodeoxycholic acid (TUDCA) significantly reduces adipogenesis of MSCs. In the present study, we examined whether TUDCA can promote osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) by regulating Integrin 5 (ITGA5) associated with activation of ERK1/2 signal pathway and thereby enhance bone tissue regeneration by reducing apoptosis and the inflammatory response. TUDCA treatment promoted in vitro osteogenic differentiation of BMMSCs and in vivo bone tissue regeneration in a calvarial defect model, as confirmed by micro-computed tomography, histological staining, and immunohistochemistry for osteocalcin. In addition, TUDCA treatment significantly decreased apoptosis and the inflammatory response in vivo and in vitro, which is important to enhance bone tissue regeneration. These results indicate that TUDCA plays a critical role in enhancing osteogenesis of BMMSCs, and is therefore a potential alternative drug for bone tissue regeneration.     

Chromosome 2q32 may harbor a QTL affecting BMD variation at different skeletal sites.

BMDs at different skeletal sites share some common genetic determinants. Using PCA and bivariate linkage analysis, we identified a QTL on chromosome 2q32 with significant pleiotropic effects on BMDs at different skeletal sites. INTRODUCTION: BMDs at the hip, spine, and forearm are genetically correlated, suggesting the existence of quantitative trait loci (QTLs) with concurrent effects on BMDs at these three skeletal sites. Consequently, it is important to identify these QTLs in the human genome and, for those implicated QTLs, it is important to differentiate between pleiotropic effects, caused by a single gene that concurrently effects these traits, and co-incident linkage, caused by multiple, closely linked, genes that independently effect these traits. MATERIALS AND METHODS: For a sample of 451 American white pedigrees made up of 4,498 individuals, we evaluated the correlations between BMDs at the three skeletal sites. We carried out principal component analysis (PCA) for the three correlated traits and obtained a major component, PC1, which accounts for >75% of the co-variation of BMDs at the three sites. We subsequently conducted a whole genome linkage scan for PC1 and performed bivariate linkage analysis for pairs of the three traits (i.e., forearm/spine BMD, hip/forearm BMD, and hip/spine BMD). RESULTS: Chromosome region 2q32, near the marker GATA65C03M, showed strong linkage to PC1 (LOD = 3.35). Subsequent bivariate linkage analysis substantiated linkage at 2q32 for each trait pair (LOD scores were 2.65, 2.42, and 2.13 for forearm/spine BMD, hip/forearm BMD, and hip/spine BMD, respectively). Further analyses rejected the hypothesis of co-incident linkage (p(0)[forearm/spine] = 0.0005, p(0)[hip/forearm] = 0.004, p(0)(hip/spine] = 0.001) but failed to reject the hypothesis of pleiotropy (p(1)[forearm/spine] = 0.35, p(1)[hip/forearm] = 0.07, p(1)[hip/spine] = 0.15). CONCLUSIONS: Our results strongly support the conclusion that chromosome region 2q32 may harbor a QTL with pleiotropic effects on BMDs at different skeletal sites.