Acceleration of bone formation during fracture healing by injectable collagen powder and human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase.

Growth factor delivered with implantable biomaterials has been used to both accelerate and ensure healing of open fractures in human patients. However, a major limitation of implantable biomaterials is the requirement for open surgical placement. Here, we developed an injectable collagen material-based bone formation system consisting of injectable collagen powder with fibril morphology and collagen triple helix conformation, and basic fibroblast growth factor (bFGF) fused to the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The affinity of the CBD towards collagen was confirmed by the results of collagen-binding assays. Moreover, the combination of the collagen binding-bFGF fusion protein (CB-bFGF) with injectable collagen powder induced bone formation at protein concentrations lower than those required for bFGF alone in mice fracture models. Taken together, these properties suggest that the CB-bFGF/collagen powder composite is a promising injectable material for bone repair in the clinical setting.

Microstructure and homogeneity of distribution of mineralised struts determine callus strength.

Non-invasive assessment of fracture healing, both in clinical and animal studies, has gained favour as surrogate measure to estimate regain of mechanical function. Micro-computed tomography (µCT) parameters such as fracture callus volume and mineralisation have been used to estimate callus mechanical competence. However, no in-depth information has been reported on microstructural parameters in estimating callus mechanical competence. The goal of this study is to use differently conditioned mice exhibiting good and impaired fracture healing outcomes and investigate the relationship between µCT imaging parameters (volume, mineralisation, and microstructure) that best estimate the callus strength and stiffness as it develops over time. A total of 99 mice with femoral fracture and intramedullary stabilisation were divided into four groups according to conditioning: wild type, NF1 knock-out, RAG1 knock-out and macrophage depleted. Animals were sacrificed at 14, 21, 28 or 35 days and µCT parameters and torsional stiffness and strength were assessed post-sacrifice. Using linear regression for all groups and time points together, torsional stiffness could be estimated with strut thickness, strut number and strut homogeneity (R² = 0.546, p < 0.0001); torsional strength could be estimated using bone mineral density, strut thickness and strut homogeneity (R² = 0.568, p < 0.0001). Differently conditioned mice that result in different fracture healing outcomes have been shown to result in varying structural, material and volumetric µCT parameters which can be used to estimate regain of bone strength. This study is the first to demonstrate that microstructure and strut homogeneity influence callus stiffness and strength.

Augmentation of autologous bone graft by a combination of bone morphogenic protein and bisphosphonate increased both callus volume and strength.

BACKGROUND AND PURPOSE: Bone morphogenic proteins (BMPs) can be used in non-unions to replace autograft. BMPs induce osteoblasts and (less well known) also osteoclasts, which can in turn be controlled by a bisphosphonate. In the present study, our aim was to improve the biological effect of autologous bone graft by adding an anabolic BMP, with or without bisphosphonates, in an open-fracture model prone to non-union. METHODS: Rat femurs were osteotomized and fixed with an intramedullary K-wire. Autograft was placed at the osteotomy, mixed with either saline or BMP-7. After 2 weeks, the rats had a single injection of saline or of a bisphosphonate (zoledronate). The rats were killed after 6 weeks and the femurs were evaluated by radiography, micro-CT, histology, and 3-point bending test. RESULTS: All fractures healed. The callus volume was doubled in the BMP-treated femurs (p < 0.01) and increased almost 4-fold in the femurs treated with both BMP and systemic zoledronate (p < 0.01) compared to autograft. In mechanical testing, the autograft group reached approximately half the strength of the contralateral, non-osteotomized femur (p < 0.001). By adding BMP to the autograft, the strength was doubled (p < 0.001) and with both BMP and systemic zoledronate, the strength was increased 4-fold (p < 0.001) compared to autograft alone. INTERPRETATION: The combination of BMP and bisphosphonate as an adjunct to autograft is superior to autograft alone or combined with BMP. The combination may prove valuable in the treatment of non-unions.

EP1(-/-) mice have enhanced osteoblast differentiation and accelerated fracture repair.

As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E(2) (PGE(2)) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro-computed tomographic (µCT), and biomechanical measures. EP1(-/-) mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1(-/-) mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1(-/-) mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE(2) receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing.

Recombinant human platelet-derived growth factor BB (rhPDGF-BB) and beta-tricalcium phosphate/collagen matrix enhance fracture healing in a diabetic rat model.

Diabetes mellitus is a common systemic disease that has been associated with poor fracture healing outcomes. The mechanism through which diabetes impairs bone regeneration is unknown. One possible mechanism may be related to either decreased or uncoordinated release of local growth factors at the fracture site. Indeed, previous studies have found reduced platelet-derived growth factor (PDGF) levels in the fracture callus of diabetic rats, suggesting that local application of PDGF may overcome the negative effects of diabetes and promote fracture healing. To test this hypothesis, low (22 microg) and high (75 ug) doses of recombinant human PDGF-BB (rhPDGF-BB) were applied directly to femur fracture sites in BB Wistar diabetic rats that were then compared to untreated or vehicle-treated animals. rhPDGF-BB treatment significantly increased early callus cell proliferation compared to that in control specimens. Low dose rhPDGF-BB treatment significantly increased callus peak torque values (p < 0.05) at 8 weeks after fracture as compared to controls. High dose rhPDGF-BB treatment increased callus bone area at 12 weeks postfracture. These data indicate that rhPDGF-BB treatment ameliorates the effects of diabetes on fracture healing by promoting early cellular proliferation that ultimately leads to more bone formation. Local application of rhPDGF-BB may be a new therapeutic approach to treat diabetes-impaired fracture healing.

A comparative histologic study of rapid three-dimensional zygomatic suture expansion osteogenesis.

OBJECTIVE: To explore the histologic changes in zygomatic suture with 3-dimensional (3D) zygomatic suture expansion osteogenesis (SEO). STUDY DESIGN: The zygomatic bones were drawn by 3D external expansion appliance, and sutures of the zygomatic were extended. Biopsy specimens of the zygomatic bone were collected after 1, 3, 5, and 8 weeks. Each specimen was stained with Triplex staining as well as hematoxylin and eosin (HE), and the histologic changes were evaluated compared with the control section. RESULTS: With the 1-week study group, there were visible fibroblasts, osteoblasts, and capillary vessels in the expanded suture tissues. The fibers were connected to the sides of suture in an orderly way. In the 3-week group, active bone formation can be seen in expanded sides. The bone trabeculae were matured and oriented in the direction of distraction in the expanded sides of suture and most of them were collagen fibers; but the tissues of both sides of expanded suture were not in order. New woven bones were found in the histologic sections of the 5-week group. In the 8-week group, reticular and elastic fibers could not be observed, and bones were formed completely. Compared with the histologic examination of the same tissue section and its change in different periods of the biopsy specimen with Triplex staining and HE staining, the method of Triplex staining showed more clearly in collagen, reticular, and elastic fiber examination. The superiority of HE staining was in showing fibrous tissue, shape of cells, and bone formation in various degrees of maturation in rapid SEO. It is suggested that the 2 staining methods might be combined in distinguishing the collagen as well as reticular fibers from fibrin in the same section simultaneously. CONCLUSIONS: Collagen fibers and new bones were observed to form rapidly in expanded sides with direct SEO. Continual affluent blood supply, integral structure of periosteum and synostoses in the suture, and rapid uniform calcification of the whole new tissues are the histologic characteristics of SEO.

Evaluation of lengthened callus by callotasis with analysis of mathematical morphology.

BACKGROUND: Callotasis has become an established option for reconstruction of congenital anomalies and after resection of bone tumors. However, fracture after removal of the lengthener remains one of the most serious complications. Mathematical morphology is an image processing technique that allows the skeletal patterns of trabecular bone to be selectively extracted as binary images from computed radiographic (CR) images. We evaluated the strength of the lengthened callus by analysis using mathematical morphology and examined the utility of this method in a biomechanical study. METHODS: Three Japanese white rabbits per time point were euthanized at the 2-, 3-, 4-, 6-, and 8-week points after completion of the lengthening and were evaluated by CR images and analysis with mathematical morphology. RESULTS: The changes of the number and continuity of trabecular bone were obtained visually and could be evaluated quantitatively. In some cases corticalization appeared to be complete in the CR images, but it was not complete on the morphological images. Such cases were found to be weaker than those in which corticalization appeared to be complete on both CR and morphological images. CONCLUSION: Analysis with mathematical morphology is useful for evaluating the lengthened callus after callotasis.

Effect of age on vascularization during fracture repair.

Age affects fracture repair; however, the underlying mechanisms are not well understood. The goal of this study was to assess the effects that age has on vascularization during fracture healing. Tibial fractures were created in juvenile (4-week-old), middle-aged (6-month-old), and elderly (18-month-old) mice. The length density and surface density of blood vessels within fracture calluses were analyzed using stereology at 7 days after fracture. The expression of molecules that regulate vascular invasion of the fracture callus was also compared among the three age groups by immunohistochemistry and in situ hybridization. At 7 days after fracture, juvenile mice had a higher surface density of blood vessels compared to the middle-aged and elderly. Hypoxia-inducible factor-1 alpha protein and transcripts of vascular endothelial growth factor were detected at 3 days postinjury in juvenile but not middle-aged and elderly mice. Stronger Mmp-9 and -13 expression was detected in fracture calluses at day 7 in the juvenile compared to the middle-aged and elderly mice. At 21 days postfracture, expression of both Mmps was more robust in the elderly than juvenile and middle-aged animals. These data indicate that age affects vascularization during fracture repair, and the changes we observed are directly correlated with altered expression of biochemical factors that regulate the process of angiogenesis. However, whether the increased vascularization is the cause or result of accelerated bone repair in juvenile animals remains unknown. Nonetheless, our results indicate that enhancing vascularization during fracture repair in the elderly may provide unique therapeutic opportunities.

Mice expressing a constitutively active PTH/PTHrP receptor in osteoblasts show reduced callus size but normal callus morphology during fracture healing.

BACKGROUND: The parathyroid hormone-/parathyroid hormone-related protein (PTH/PTHrP) receptor plays a crucial role in endochondral bone formation and possibly also in fracture healing. Patients with Jansen's metaphysial chondrodysplasia (JMC) have a gain-of-function mutation in the PTH/PTHrP receptor. Transgenic mice expressing JMC PTH/PTHrP receptor mutants in osteoblasts are characterized by increased trabecular bone formation and reduced osteoblastic activity at periosteal sites. We have analyzed the bone phenotype and studied the fracture healing process in this model. METHODS: We performed bone density analysis of tibiae from 17-week-old transgenic mice and controls. Also, tibial fractures were produced in 14-week-old mice. Fracture healing was examined by radiographic and histological analysis. RESULTS: Transgenic mice had a lower total bone mineral content (BMC), by a factor of one-third. The changes were bone compartment-specific with an increase in trabecular bone volume and a decrease in cortical thickness. The calluses in the transgenic mice were smaller, with a reduction in BMC and mean cross-sectional area by a factor of one-half. Despite the smaller size, however, the morphology and progression through the healing process were similar in both transgenic and wild-type littermates. INTERPRETATION: We conclude that the constitutively active PTH/PTHrP receptor has compartment-specific effects on bone formation when expressed in osteoblasts. During fracture healing, however, both the periosteal and the endochondral processes are activated, leading to fracture healing that is temporally and morphologically normal, although the callus tissue is less prominent.

Alveolar distraction osteogenesis for bone augmentation of severely atrophic ridges in 10 consecutive cases: a histologic and histomorphometric study.

BACKGROUND: This study analyzed bone healing in surgically osteodistracted maxillary and mandibular ridges histologically and histomorphometrically at two different times to determine the best time to insert dental implants. METHODS: Ten consecutive patients with severe maxillary (two patients) or mandibular (eight patients) atrophy underwent surgical osteodistraction with an extraosseous distractor. Seven days after the surgery, the distractor was activated at a rate of 1 mm/day until achieving the planned bone lengthening. The distractor was removed after a consolidation period of 70 days. Bone biopsies were obtained at implant insertion: 70 days after the end of distraction on the day of distractor removal in six patients (group A) or 180 days afterwards in four patients (group B). The biopsies were evaluated histologically and histomorphometrically to measure the osteocyte lacunar area (OLA). RESULTS: The histologic and histomorphometrical analysis of the distracted bone 70 days after the end of distraction showed well-organized lamellar bone. At 180 days, the bone was more compact and mature; the mineralization of the matrix was greater; and an increased, but small, amount of marrow space was evident (35% versus 45%). The mean OLA was 80.11 +/- 27.59 microm2 in group A and 70.4 +/- 33.58 microm2 in group B. The difference between the two biopsy groups was not significant (P = 0.315). CONCLUSION: The results of this study showed that there was definitely similar bone formation in the distracted area for both healing periods, and placing implants clinically worked in both of these time periods in the limited number of cases observed.

Enhanced fracture and soft-tissue healing by means of anabolic dietary supplementation.

BACKGROUND: Malnutrition is common in hospitalized injured patients. It contributes to delayed fracture-healing and increased morbidity. However, relatively little attention has been directed toward nutritional strategies for augmenting musculoskeletal recovery after a fracture. This animal study was designed to examine the effects of dietary protein intake and the role of conditionally essential amino acids in muscle and bone-healing after a fracture. METHODS: One hundred adult male rats were used. Ten rats served as controls and received a 15% protein diet throughout the study. The remaining ninety rats received a 6% protein diet for five weeks to induce protein malnutrition. The rats underwent intramedullary nailing and closed midshaft fracture of one femur. After the fracture, they were separated into three isocaloric dietary groups. Group P6 received a diet with 6% protein; Group P15, a diet with 15% protein; and group P30, a diet with 30% protein with conditionally essential amino acids. At two, four, and six weeks after surgery, ten animals from each group were killed and the femora were evaluated with dual x-ray absorptiometry, histomorphometric assessment of callus, and torsional testing. The quadriceps muscles were analyzed for total mass, total protein content, and for mRNA expression of insulin-like growth factor-1 (IGF-1), IGF-2, IGF receptors, actin, myosin, and vascular endothelial growth factor (VEGF). RESULTS: The P30 group demonstrated elevations in albumin, body mass, muscle mass, total protein content of muscle, and bone mineral density in the fracture callus compared with the P6 diet group at six weeks (p < 0.05). Molecular analysis of muscle revealed that IGF-1, IGF-2, IGF receptors, myosin, actin, and VEGF gene expression were significantly (p < 0.001) higher in the P6 group compared with the P30 group. Biomechanical testing of the femora, however, showed no significant differences. CONCLUSIONS: Dietary supplementation with conditionally essential amino acids in malnourished animals had anabolic effects on bone mineralization, body mass, and muscle mass.

Direct adenoviral transfer of bone morphogenetic protein-2 cDNA enhances fracture healing in osteoporotic sheep.

Osteoporosis, a major public health burden, is associated with increased fracture risk. Fracture healing in osteoporosis is delayed, with reduced callus formation and impaired biomechanical properties of newly formed bone leading to high risk of fixation failure. Adenoviral gene transfer of bone morphogenetic protein-2 (BMP-2) has been shown to enhance fracture healing. This study evaluated the ability of gene transfer to enhance bone healing in osteoporosis. An established sheep model of osteoporosis with well-characterized alterations in fracture healing was used. Osteotomies were created surgically in the tibias of adult female sheep and monitored for 8 weeks, using radiographic, biomechanical, and histological methods. For pilot experiments, primary ovine osteoblasts and mesenchymal stem cells were transduced with a recombinant adenovirus carrying BMP-2 cDNA (Ad.BMP-2). Large increases in alkaline phosphatase production and mineralization confirmed the ability of human BMP-2 to stimulate osteoblastic differentiation in sheep. In vivo bending stiffness measurements during fracture healing as well as ex vivo torsional stiffness measurements demonstrated stiffer callus tissue after treatment with Ad.BMP-2. The differences were found mainly in the early fracture-healing period. Computed tomography demonstrated that animals receiving the BMP-2 cDNA had larger cross-sectional callus area and higher callus density. Histological examination of the tibias confirmed enhanced callus formation. Direct, local adenoviral delivery of an osteogenic gene thus led to enhanced healing of fractures in an ovine model of osteoporosis. These promising data encourage the further development of genetic approaches to enhance bone healing in patients suffering osteoporosis-associated fractures.

Do serological tissue turnover markers represent callus formation during fracture healing?

Serological parameters of bone and fibrous tissue turnover were demonstrated to monitor the course of fracture healing. The aim of this study was to evaluate the correlation between the serological parameter levels during fracture healing and callus development in a standardised ovine model of fracture healing. Two years old female sheep received a standardised 3 mm tibial bone defect stabilised by an external fixator. The serological levels of the C-terminal propeptide of procollagen type I (PICP), bone specific alkaline phosphatase (bALP), total alkaline phosphatase (tALP), osteocalcin, tartrate-resistant acid phosphatase (TRAP), calcium, phosphate and the N-terminal peptide of procollagen type III (PIIINP) were observed over a 9-week healing period. The course of fracture healing was monitored radiographically, and the callus composition was evaluated histologically at 2, 3, 6 and 9 weeks post-surgery. The serological results were compared with an untreated control group. Additionally, the maximum values during healing were compared with juvenile values to gauge the level of the serological response. The histological and radiographical results demonstrated callus formation without complications. All serological parameters showed broad inter-individual variations, and the response to the standardised fracture scenario was strongly individual. Maximum values during fracture healing did not reach the juvenile levels. The fractured as well as the control animals showed significant changes in the parameter levels. No correlations were observed between the histological course of healing and the course of bone formation markers whilst the TRAP level was reduced during bony callus formation. The PIIINP level increased when the amount of soft callus tissue decreased during healing. The observed bone formation markers were not suitable as general markers to detect the course of fracture healing, whilst PIIINP was able to reflect soft callus degradation.

Trabecular organization in mandibular osteodistraction in growing and maturing rats.

PURPOSE: Our goal was to investigate the trabecular organization of the distraction regenerate during various consolidation phases and as a result of various distraction rates. MATERIALS AND METHODS: One hundred ninety-two growing and maturing rats (96 each) received unilateral mandibular osteotomies and distraction device placement. They were randomly allocated into 4 distraction rate groups (0, 0.2, 0.4, and 0.6 mm/day for 5 days) after a 3-day latency. Eight rats from each rate group were sacrificed at early (10 days), mid (24 days) and late (38 days) consolidation time points. Harvested hemimandibles were embedded in micro-bed resin, sectioned sagittally at 10 mum thickness and stained using the Von Kossa method. The histologic images were captured and processed using Adobe Photoshop (Version 7.0; Adobe Systems Inc, San Jose, CA). Custom-made software (MacAzimuth; written by Prof. J.M. Rensberger, University of Washington) was further used to analyze the orientation (anisotropy and angle distribution) and mass (density and thickness) of trabecular structures in the regenerates. RESULTS: Trabecular orientation significantly differed at the mid-consolidation time point with less anisotropy ( P < or = .000 to .023) and narrower angular distribution ( P < or = .007 to .021). Trabecular mass increased significantly from early to mid-consolidation ( P < or = .000 to .002). Growing rats showed greater trabecular organization than maturing rats did at the same time points ( P < or = .000 to .009). The trabecular organization parameters were associated significantly with bone mineral density and microdensity ( P < or = .000 to .044). CONCLUSION: The peak change in orientation occurred around the mid-consolidation with a greater increase in trabecular mass. Growing rats showed a greater capacity for trabecular organization at earlier time points. However, distraction rate did not have an effect on trabecular organization. These results suggest that trabecular organization can be used as an important indicator to evaluate bone maturation and quality in the distraction regenerate.

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression.

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages.

Acceleration of callus maturation using rhOP-1 in mandibular distraction osteogenesis in a rat model.

The aim of this study was to assess if the application of rhOP-1 induces accelerated consolidation of the callus in mandibular distraction osteogenesis. In seven adult Wistar rats a bilateral osteotomy of the horizontal ramus of the mandible was performed in the molar region and a custom designed distractor was mounted to the mandible. With a rate of 0.7 mm per day the device was activated bilaterally after the seventh postoperative day. After seven days of distraction two times 50 microg rhOP-1 were injected on two subsequent days directly into the callus. The contralateral side received an injection of placebo solution. The animals were killed four weeks after the end of distraction. A three-point bending test revealed a significantly higher strength of the distracted mandible in the rhOP-1 side (66.3 N vs. 30.4 N, P=0.034, paired t-test). Undecalcified histological sections were examined using microradiography and fluorescence microscopy after sequential intravital polychromic labelling. A continuous bony bridging was seen in all rhOP-1 sites and in none of the control sites. The data indicate that rhOP-1 may be an option to accelerate callus maturation in mandibular distraction osteogenesis.

Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34).

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1-34) [PTH(1-34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1-34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 microg/kg of PTH(1-34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH-treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)-positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH-treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COLlA1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH-treated group and accompanied by enhanced expression of insulin-like growth factor (IGF)-I mRNA during the early stages of healing (days 4-7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14-21) despite a lack of up-regulation of IGF-I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1-34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low-dose PTH(1-34) to enhance fracture healing are indicated.

Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing.

Fracture healing is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Recent evidence for the role of tumor necrosis family members in the coupling of cellular functions during skeletal homeostasis suggests that they also may be involved in the regulation of skeletal repair. The expression of a number of cytokines and receptors that are of functional importance to bone remodeling (osteoprotegerin [OPG], macrophage colony-stimulating factor [M-CSF], and osteoprotegerin ligand [receptor activator of NF-kappaB ligand (RANKL)]), as well as inflammation (tumor necrosis factor alpha [TNF-alpha] and its receptors, and interleukin-1alpha [IL-1alpha] and -beta and their receptors) were analyzed over a 28-day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M-CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF-alpha, lymphotoxin-beta (LT-beta), IL-1alpha, and IL-1beta showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF-alpha receptors (p55 and p75) and the IL-1RII receptor showed identical patterns of expression to their ligands, while the IL-1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF-alpha and IL-1alpha expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF-alpha expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M-CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF-alpha and IL-1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.

Análisis biomecánico del callo óseo en fracturas experimentales tratadas con xenoinjerto de hueso marino (sepsia officinalis) / Biomechanical analysis of bone callus in experimental fractures treating with marine bone (sepsia officinialis) xenerograft

El presente trabajo se realizó con la finalidad de estudiar una nueva variante de xenoinjerto (hueso de sepsia officinalis), con el fin de demostrar la integración estable, conferiéndole características biomécanicas similares a las del hueso sano en defectos óseos experimentales creados en el 1/3 medio del fémur de animales de experimentación (conejos). La investigación se llevó a cabo en un período de 4 meses (mayo - agosto del 2000), durante el cual se realizó estudios clínicos y radiológicos que nos permitieron apreciar la integración estable del injerto de hueso marino. Al poseer confirmación clínico - radiológico de esta integración se procedió a sacrificar los animales de experimentación con el fin de obtener las muestras necesarias para realizar los estudios pertinentes. Posteriormente mediante el uso de una prensa electromecánica se procedió a realizar una serie de estudios biomecánicos de precisión que permiten conocer las características propias del callo desarrollado posterior a la implantación del hueso marino. Dicho estudio demostró que en aquellos especímenes tratados con hueso marino aumentó la elasticidad y por tanto la ductibilidad del hueso tratado. En cuanto a la resistencia del callo postimplante, este demostró poseer características similares a las del hueso sano, lo que nos permitió inferir que el implante de Sepsia Officinalis además de integrarse totalmente adquirió las características de un hueso sano o de neoformación del huésped. Se concluye que esta nueva alternativa de xenoinjerto tiene amplias posibilidades de aplicación en la cirugía ortopédica y traumatológica, así como en otras especialidades médicas tales como la odontología y la cirugía plástica, siendo además económica, de fácil acceso, manejo y sin incremento de la morbilidad

Tissue differentiation and cytokine synthesis during strain-related bone formation in distraction osteogenesis.

To investigate the contributions of various cytokines that are involved in mechanically related bone formation, we applied defined uniaxial strains in a rabbit model of mandibular elongation and examined the regenerating bone during early stages of dist raction osteogenesis by histomorphometry. We also measured serum concentrations of various cytokines during the distraction. Cell proliferation and differentiation indices correlated significantly (P<0.001) with the extent of load application. Serum concentrations of insulin-like growth factor-1 (IGF-1) decreased after osteotomy whereas transforming growth factor beta1 (TGFbeta1) showed a postoperative increase. Prostaglandin E2 (PGE2) concentrations were constant throughout the experimental period. Collagen degradation decreased slightly postoperatively and increased in samples exposed to higher magnitudes of strain. Our data show that it is the magnitude of mechanical strain that decides tissue response by a characteristic cell proliferation and differentiation. The operative trauma leads to inverse changes in serum concentrations of TGFbeta1 and IGF-1, thereby promoting the recruitment of osteoblastic precursor cells as well as collagen matrix synthesis.