Early Stages Of Fracture Healing Research Articles

Early changes of fracture healing on MRI

Objective To observe MRI presentations of fracture healing process in the early stage.Methods Six New Zealand rabbits were used in the experiment. A union model of long oblique fracture of 1 mm in bone defect and 10 mm in vertical length was created on the right radius. A model of 10 mm bone defect was created on the left radius. X-ray and MRI examinations were performed in 1 w, 2 w, 4 w, 6 w, 8 w, 12 wafter the surgery and CT examination was performed in 4 w, 12 w after the surgery. The imaging presentations at the fracture sites were observed on T1, T2, FS-T2, PD, FS-PD sequences. Results The soft tissue around the fracture site presented high signals on T2, FS-T2, PD, FS-PD sequences in the early stage of fracture healing. The signals decreased as time went on, but turned intermediate at 6 w after surgery. The signals of bone marrow turned from high to intermediate on T1, T2 and PD sequences early after the surgery,but turned high again with the healing of medullary cavity. On FS-T2 and FS-PD sequences, bone marrow presented intermediate signals mixed with high signals, but the high signals subsided as time went by. Early changes of callus formation could be observed by MRI. Callus formation at the site of fracture showed intermediate signals in FS-T2 and FS-PD images obtained at 2 w after the surgery in the fracture union model. The changes above could not be observed in the bone defect model. Conclusions Callus formation may be predicted earlier by MRI than by X ray, especially on FS-T2 and FS-PD sequences. The MRI presentations of early fracture healing are to be confirmed by pathological examinations. Key words: Fracture healing; Magnetic resonance image; Animal experimentation

Local transplantation of human multipotent adipose-derived stem cells accelerates fracture healing via enhanced osteogenesis and angiogenesis

Adipose tissue is one of the promising sources of multipotent stem cells in human. Human multipotent adipose-derived stem (hMADS) cells have recently been isolated and showed differentiation potential into multiple mesenchymal lineages in vitro and in vivo. On the basis of these evidences, we examined the therapeutic efficacy of hMADS cells for fracture healing in an immunodeficient rat femur non-union fracture model. Local transplantation of hMADS cells radiographically and histologically promoted fracture healing with significant improvement of biomechanical function at the fracture sites compared with local transplantation of human fibroblasts (hFB) or PBS administration. Histological capillary density and physiological blood flow by laser Doppler perfusion imaging were significantly greater in hMADS group than hFB and PBS groups. Expressions of intrinsic (rat) bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and angiopoietin-1 in peri-fracture tissue were upregulated in hMADS group than other groups. In addition, presence of BMP-2 or VEGF activated the proliferation and migration of hMADS cells in vitro. These results indicate that hMADS cells stimulate the interaction between the transplanted cells and the resident cells stronger than other cells, and they promote fracture healing more effectively. Furthermore, immunohistochemistry for human-specific antibodies revealed direct differentiation of hMADS cells into osteoblasts or endothelial cells in newly formed callus or vasculature, respectively. RT-PCR for human-specific primers for osteogenic/endothelial markers also disclosed osteogenic and vasculogenic plasticity of the transplanted hMADS cells at the early stage of fracture healing. The present results suggest that transplantation of hMADS cells may become a useful strategy for cell-based bone regeneration in the future clinical setting.

Intermittent low-dose administration of recombinant human parathyroid hormone (1-34) promotes the expression of Runx2 during early stage of fracture healing

To study the correlation of intermittent low-dose administration of recombinant human parathyroid hormone (1-34) [rhPTH (1-34)] and the expression of Runx2 during early stage of fracture healing. Sixty 2-month old male Sprague-Dawley rats underwent close unilateral femoral fracture and intramedullary nail fixation, and then were randomly divided into 2 equal groups: treatment group undergoing subcutaneous injection of rhPTH (1-34) 10 microgxkg(-1)xd(-1) immediately after the operation, and control group undergoing subcutaneous injection of normal saline of the same dose. Six rats from each group were killed with their bilateral femurs taken out on days 2, 4, 7, 14, and 21 after the operation to undergo X-ray photography Tissue RNA and protein were extracted from the bone tissues and the levels of Runx2 mRNA and protein expression were evaluated through real time quantitative PCR and Western-blotting. Blood samples were collected from the abdominal aorta before the rats were killed to undergo detection of serum calcium, phosphorus, and alkaline phosphatase (AKP). The Runx2 mRNA levels in the fractured femurs of the rhPTH (1-34) group on days 14 and 21 after the operation were 2.6 and 3.8 times as those of the control group respectively (both P<0.05). The Runx2 protein levels in the fractured femurs of the rhPTH (1-34) group on days 14 and 21 after the operation were significantly higher than those of the control group respectively too. Since day 14 fracture healing was seen, and the status of fracture healing was better in the rhPTH (1-34) group than in the control group on days 14 and 21. The levels of serum calcium ion on days 14 and 21 of the rhPTH (1-34) group were both significantly higher than those of the control group (both P<0.05). There were not significant differences in the serum levels of AKP, osteocalcin and collagen type Iat any time point between the 2 groups. Intermittent low-dose administration of rhPTH (1-34) up-regulates the levels of osteogenesis-specific Runx2 mRNA and protein expression to accelerate the early stage fracture healing during the early stage.

Patients with long bone fracture have altered Caveolin-1 expression in their peripheral blood mononuclear cells

Fracture triggers a cascade of systemic and local responses including inflammatory mediator signaling, chemotaxis, osteogenic cell recruitment, differentiation and proliferation at the fracture site. Early signaling between immune cells and repair cells in fracture repair is not well understood. Caveolin-1, a 21-24 kDa membrane protein plays key roles in transmembrane signaling. This study was to investigate the expression of caveolin-1 in human peripheral blood mononuclear cells (PBMNCs) following long bone fracture. PBMNCs were obtained from healthy volunteers or fracture patients at three time points following fracture by density-gradient-centrifugation procedure. Caveolin-1 gene expression and protein characterization was examined by semi-quantitative RT-PCR, immunocytochemistry and Western blot analysis. Caveolin-1 mRNA and protein was expressed at low levels in the PBMNCs of non-fracture samples. In contrast, caveolin-1 expression was greatly increased in the PBMNCs of fracture patients 9-12 days and reduced at 16-21 days following long bone fracture. The identification of caveolin-1 in PBMNCs and osteoblasts makes this cellular domain a new focus for further investigation. We speculate that caveolin-1 expression in PBMNCs and osteoblasts play an important role in signal transduction during the early stages of fracture healing and may be an indicator for normal or abnormal fracture repair.

Bax deficiency in mice increases cartilage production during fracture repair through a mechanism involving increased chondrocyte proliferation without changes in apoptosis

This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had ∼ 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and “Bik-like” pro-apoptotic gene increased ∼ 1.5-fold and ∼ 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2α1 and 9α1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair.

Pause Insertions During Cyclic In Vivo Loading Affect Bone Healing

Fracture repair is influenced by the mechanical environment, particularly when cyclic loads are applied across the fracture site. However, the specific mechanical loading parameters that accelerate fracture healing are unknown. Intact bone adaptation studies show enhanced bone formation with pauses inserted between loading cycles. We hypothesized pause-inserted noninvasive external loading to mouse tibial fractures would lead to accelerated healing. Eighty mice underwent tibial osteotomies with intramedullary stabilization and were divided into four loading protocol groups: (1) repetitive loading (100 cycles, 1 Hz); (2) pause/time-equivalent (10 cycles, 0.1 Hz); (3) pause/cycle-equivalent (100 cycles, 0.1 Hz); and (4) no load control. Loading was applied daily for 2 weeks. Healing was assessed using histology, biomechanical bending tests, and microcomputed tomography. The pause-inserted, cycle-equivalent group had a greater percentage of osteoid present in the callus cross-sectional area compared with no-load controls, indicating more advanced early healing. The pause-inserted, cycle-equivalent group had a failure moment and stiffness that were 37% and 31% higher than the controls, respectively. All three loaded groups had smaller overall mineralized callus volumes than the control group, also indicating more advanced healing. At an early stage of fracture healing, pause-inserted loading led to more histologically advanced healing.

Circulating cells with osteogenic potential are physiologically mobilized into the fracture healing site in the parabiotic mice model

Based on the accumulating evidence of osteogenic cells present in the systemic circulation, we hypothesized that circulating osteogenic connective tissue progenitors (CTPs) home to fracture site and contribute to skeletal repair. Parabiotic animals were formed by surgically conjoining transgenic mice constitutively expressing green fluorescent protein (GFP) in no erythroid tissue and syngeneic wild-type mice. After 3 weeks parabionts, equilibrium in blood chimerism between partners was established. A transverse fibular fracture was made in the contralateral hind limb of the conjoined wild-type partner. The contribution of circulating cells to the fracture callus was assessed based on analysis of GFP+ cells and co-localization of alkaline phosphatase (AP) staining nonfracture and at 1, 2, 3, and 4 weeks after fracture. Histomorphometric analysis at the fracture site showed significant increase of GFP+ cells after 2 (5.4%) and 3 (5.6%) weeks compared to nonfractured controls (1.7%). Of the GFP+ cells, percentage of the cells expressing AP activity at 1 (37.4%) and 2 (85.3%) weeks postfracture time was statistically higher than that in nonfractured controls (10.8%). The rate of mobilization of circulating osteogenic CTPs to fracture callus was also examined using 1 week parabionts at week 0-1 and week 1-2 postfracture. There was significant increase of GFP+/AP+ cells from week 0-1 (0.1%) and week 1-2 (1.8%). These data indicate that circulating osteogenic CTPs are mobilized to fracture site and contribute to osteogenesis in the early stage of fracture healing.

Callus mineralization and maturation are delayed during fracture healing in interleukin-6 knockout mice

IL-6 is a pleiotropic cytokine involved in cell signaling in the musculoskeletal system, but its role in bone healing remains uncertain. The purpose of this study was to examine the role of IL-6 in fracture healing. Eight-week-old male C57BL/6 and IL-6 −/− mice were subjected to transverse, mid-diaphyseal osteotomies on the right femora. Sacrifice time points were 1, 2, 4, or 6 weeks post-fracture ( N = 14 per group). Callus tissue properties was analyzed by microcomputed tomography (micro-CT) and Fourier transform infrared imaging spectroscopy (FT-IRIS). Cartilage and collagen content, and osteoclast density were measured histologically. In intact unfractured bone, IL-6 −/− mice had reduced crystallinity, mineral/matrix ratio, tissue mineral density (TMD), and bone volume fraction (BVF) compared to wildtype mice. This suggests that there was an underlying deficit in baseline bone quality in IL-6 −/− mice. At 2 weeks post-fracture, the callus of IL-6 −/− mice had reduced crystallinity and mineral/matrix ratio. These changes were less evident at 4 weeks. At 2 weeks, the callus of the IL-6 −/− mice had an increased tissue mineral density (TMD), an increased cartilage and collagen content, and reduced osteoclast density compared to these parameters in wildtype mice. By 4 and 6 weeks, these parameters were no longer different between the two strains of mice. In conclusion, IL-6 −/− mice had delayed callus maturity, mineralization, and remodeling compared with the callus of the wildtype mice. These effects were transient indicating that the role of IL-6 appears to be most important in the early stages of fracture healing.

Fracture repair: Modulation of fracture-callus and mechanical properties by sequential application of IL-6 following PTH 1–34 or PTH 28–48

Fracture healing presents a sequence of three major stages: inflammation and granulation tissue formation, callus formation and remodeling. Our working hypothesis was that fracture-repair might be enhanced by stimulating proliferation of chondrocytes and osteoblasts in the early stages of fracture healing followed by sequential acceleration of the remodeling process. In the present study we employed a novel device developed by us implementing a standardized fracture in rat tibiae. We investigated the effect of PTH 28–48 or PTH 1–34 alone or in sequence combination with IL-6 together with its soluble receptor (IL-6sR) on fracture repair. PTH 28–48 or PTH 1–34 was applied locally into the hematoma of fractures on days 4, 5 and 6 and IL-6+ its soluble receptor on days 7, 9, and 11. Post-fracture callus volume as measured 14 days post-fracture was increased significantly only by PTH 1–34 (20%; P<0.01). When one of the PTH fragments and IL-6+IL-6sR were applied sequentially callus volume was increased significantly (33%; P<0.01). X-rays radiography at 5 weeks post-fracture showed enlarged callus volume following treatment by either PTH fragments alone, and complete union following the sequential injection of both PTH fragments and IL-6+IL-6sR, only. Only the combination of one of the PTH fragments with IL-6+IL-6sR, as measured 6 weeks post-fracture by three point bending, changed dramatically the quality of the regenerating bone as presented by a 300% increase in mechanical resistance when PTH 1–34 was combined and 200% when PTH 28–48 was combined relative to vehicle-treated fractured bones. We conclude that the sequential application of IL-6+IL-6sR with both PTH fragments has the potential of enhancing fracture healing in long bones and should be further explored in preclinical and in clinical studies.

Expression of EDA+ and EDB+ fibronectin splice variants in bone

The extracellular matrix component fibronectin (fn) has fundamental functions in cell attachment, differentiation, proliferation, and migration. Isoforms of cellular fibronectin, named EDA+ fibronectin or embryonal EDB+ fibronectin, are generated by alternative splicing of its mRNA precursors. Little is known about the expression of EDA+ and EDB+ fibronectin splice variants in human bone. The aim of this study was to investigate the expression pattern of fibronectin splice variants in bone cell lines and in different human bone tissue samples (mature bone, early stages of fracture healing, hypotrophic nonunion, osteosarcoma). Analysis was done by immunostaining with recombinant and monoclonal antibodies, qualitative RT–PCR and LightCycler-based real-time quantitative RT–PCR assay. In osteoblast and osteosarcoma cell lines, abundant expression of EDA+ and EDB+ fibronectin was found in immunocytochemistry. High transcription levels of both splice variants mRNA were seen in quantitative RT–PCR in osteosarcoma cell lines. In mature bone, EDA+ and EDB+ were not detectable in immunohistochemistry. Transcription of mRNA in both splice variants was absent in these samples. Early stages of fracture healing and osteosarcoma cell samples exhibited extensive staining for EDA+ and EDB+ fibronectin, and high mRNA levels were found. Both osteosarcoma and bone fracture healing tissue expressed high mRNA levels of the fibronectin splice variants independent of benign or malignant behavior. Low level of EDA+ fibronectin mRNA transcription and focal immunohistochemical staining of EDA+ fibronectin was found in hypotrophic nonunions, whereas EDB+ fibronectin was not detected by immunohistochemistry and qualitative or quantitative PCR. EDA+ fibronectin was found in granulation tissue-forming processes in bone independent from bone-forming activity. EDB+ fibronectin was seen only in high-turnover new osteoid-forming processes like early stages of fracture healing and osteosarcoma and was absent in low-turnover processes like mature bone and hypotrophic nonunion. Both EDA+ and EDB+ fibronectin mark active processes in bone without differentiation between malignant or benign activity. In conclusion, EDA+ and EDB+ fibronectin splice variants are strong markers for active fibrogenetic and osteoid-forming processes in human bones.

Gene expression of periostin in the early stage of fracture healing detected by cDNA microarray analysis

Gene expression of periostin in the early stage of fracture healing detected by cDNA microarray analysis

Experimental study on the effect of mechanical stimulation on the early stage of fracture healing.

In an attempt to ascertain the effects of mechanical stimulation on callus in the early stage of bone fracture healing, a tibial fracture was induced in rats and mechanical stimulation applied to the fractures. The callus was then measured quantitatively, while the fractures were analyzed both radiographically and histologically. Following the induction of a closed transverse fracture in the tibia, external anchors were applied and the rats raised by suspending the fractured leg. The rats were divided into two main groups: a Stimulation Group (S Group) and a Control Group (C Group) without the application of any mechanical stimulation. The S Group was further divided into the following three subgroups: an axial compression group (Sc Group) receiving stimulation in the positive direction; an axial distraction group (Sd Group) receiving stimulation in the negative direction; and an axial dynamization group (Sdy Group) receiving stimulation in both directions alternately. For mechanical stimulation, 1.4-N sine waves were applied continuously for 30 minutes a day, three times a week, starting 2 days after fracture-inducing surgery. At 3, 7, and 14 days after surgery, transverse sections of each fractured bone sample were prepared. At 14 days after surgery, each transverse section was divided into two peripheral and central regions to permit calculation of the area ratio of callus. Radiographically, no marked differences were observed among the groups; histologically, differences were seen 7 days after surgery, suggesting that mechanical stimulation facilitated bone healing soon after surgery. At 14 days after surgery, the amount of callus for the C Group was less than that for all three stimulation groups. In the C Group, the amount of callus in the peripheral region was greater than in the central region, and in the Sc Group, the results were the same: callus in the peripheral region was greater than in the central region. In the Sd Group, callus was greater in the central region than in peripheral regions. In the Sdy Group, favorable callus was observed in both the central and peripheral regions. These findings suggest that axial compression facilitates callus primarily in the peripheral region, while axial distraction facilitates callus primarily in the central region. When axial compression and distraction were alternated (dynamization), callus was significantly facilitated in both the central and peripheral regions. Of the three axial stimulation techniques, dynamization was the most effective in facilitating callus in the early stage of bone fracture healing.

Gene expression of periostin in the early stage of fracture healing detected by cDNA microarray analysis

To comprehensively evaluate gene expression in the early stage of fracture healing, we used a cDNA microarray with 2304 cDNA clones derived from an oligo-capped mouse embryo library. Closed mid-diaphyseal fractures were created in mouse tibiae and expression profiles were analyzed 3 days after fracture. Six genes were up-regulated in comparison to those in unfractured bones and these included three genes previously identified but never shown to be present in fractures, periostin, calumenin, and FHL-1. Cloning of these genes has been completed but their expression pattern and function during fracture healing and bone formation remain to be elucidated. Up-regulation of the six genes was reconfirmed by semi-quantitative RT-PCR analysis. Spatial and temporal expression of one of the newly identified fracture-induced genes, periostin, was analyzed using in situ hybridization, because it displayed the highest up-regulation ratio. A signal for periostin was detected in undifferentiated mesenchymal cells and immature preosteoblastic cells in the periosteal tissues between days 3 and 14 after fracture. Northern analysis showed that periostin gene expression rapidly increased by day 3, reached a peak on day 7, and declined by day 14. These findings suggest that periostin is a specific marker for preosteoblasts and may play an important role in periosteal callus formation during the early stage of fracture healing.

Determination of the complete cDNA sequence of rat type II collagen and evaluation of distinct expression patterns of types IIA and IIB procollagen mRNAs during fracture repair in rats

AbstractElucidating the molecular mechanisms that underlie fracture healing is crucial to understanding and devising strategies for the management of fractures, especially those associated with a pathological condition such as diabetes or old age. Cartilage formation, and therefore the expression of type II collagen by chondrocytes, is a critical step in fracture healing. Two forms of type II collagen, IIA and IIB, are known to be produced by alternative splicing of the α1 (II) procollagen gene. We have followed the patterns of expression of these two forms of type II collagen to determine the nature of chondrocyte recruitment during fracture healing. First, we sequenced the rat collagen type II cDNA to design the primers. Second, using a competitive quantitative reverse transcription-mediated polymerase chain reaction, we provide evidence that (1) there is a basal level of type IIA collagen expression during the early stages of fracture healing; (2) transient but sharp up-regulation of IIA expression occurs concomitant with chondrogenesis and endochondral ossification; and (3) type IIB collagen is the predominant mRNA variant expressed at virtually all times during fracture repair.

Decreased platelet derived growth factor expression during fracture healing in diabetic animals.

Animal model experiments have suggested that diabetes inhibits cell proliferation during fracture healing. Immunohistochemical analysis of proliferating cell nuclear antigen revealed significant reductions in cellular proliferation rates in the fracture callus of spontaneously diabetic BB Wistar rats as compared with healthy BB Wistar rats. Because platelet derived growth factor is associated with the early stage of fracture healing, it was hypothesized that diabetes causes decreased platelet derived growth factor expression during the early phase of fracture healing with a concomitant decrease in cell proliferation. Midshaft femur fractures were created in healthy and spontaneously diabetic BB Wistar rats and analyzed at Days 2, 4, and 7 after fracture for expression of platelet derived growth factor. Immunohistochemistry showed decreased localization of platelet derived growth factor in early diabetic fracture callus compared with healthy controls. Platelet derived growth factor messenger ribonucleic acid levels, as determined by reverse transcription and polymerase chain reaction, also were decreased in early diabetic fractures compared with healthy controls. Therefore the decreased cell proliferation rates associated with diabetic fracture healing are consistent with decreased platelet derived growth factor levels and suggest a causal relationship. These results suggest that diabetes is affecting the early phase of fracture healing by inhibiting cell proliferation through decreasing expression of platelet derived growth factor.

In vivo new bone formation by direct transfer of adenoviral-mediated bone morphogenetic protein-4 gene

Previous studies have demonstrated that bone morphogenetic protein-4 (BMP4) could participate in vivo endochondral ossification and is one of the main local contributing factors in the early stage of fracture healing. To investigate the effectiveness of BMP4 gene transfer, we constructed an adenoviral vector, Ad-BMP4, and evaluated its osteoinduction activity both in vitro and in vivo. In vitro study suggested that this vector could efficiently transduce mouse myoblast C2C12 cells and produce osteogenic BMP4 protein, as confirmed by immunofluorescence analysis and alkaline phosphatase activity assay. For in vivo study, Ad-BMP4 was directly injected into the hind limb muscles of male athymic nude rats. Visible new bone formation under X-ray films could be detected as early as three weeks post-injection. The bone tissue was further analyzed by histological staining and revealed a typical remodeled bone structure. In conclusion, this study is the first to establish the feasibility of adenovirus-based BMP4 gene therapy for bone regeneration.

Prevention of fracture healing in rats by an inhibitor of angiogenesis

Angiogenesis is considered essential to fracture healing, but its role in the healing process remains poorly understood. Angiogenesis inhibitors, which block new blood vessel formation by specifically targeting vascular cells, are currently under development for use in cancer chemotherapy, and are potentially powerful tools for defining the consequences of angiogenic impairment on fracture healing. In this study, we directly tested the effects of the angiogenesis inhibitor TNP-470 on the healing of closed femoral fractures in an established rat model system. Beginning 1 day after fracture, animals received either angiogenesis inhibitor at a therapeutically effective antitumor dose, or a weight-adjusted amount of carrier vehicle. The progress of fracture healing was assessed at weekly intervals for 21 days by radiography and histology; functional assessment was carried out at day 24 by biomechanical testing. By all three criteria, treatment with the angiogenesis inhibitor completely prevented fracture healing. Formation of both callus and periosteal woven bone were suppressed, indicating that both the intramembranous and endochondral pathways of osteogenesis were affected. The resulting tissue resembled “atrophic nonunions” often seen clinically in cases of failed fracture healing, but rarelly achieved in animal models. These results show that angiogenesis is essential to very early stages of fracture healing, and suggest this model system may be useful for understanding the mechanisms underlying fracture nonunions due to vascular impairment. Finally, the data raise the possibility that impairment of fracture healing may be an adverse effect of clinical treatments with antiangiogenic drugs.

Percutaneous and limited open reduction of intra-articular distal radial fractures.

Fluoroscopy monitors intra-articular distal radial fracture reduction and stabilisation. The reduction is guided by traction, ligamentotaxis and manipulation, and when necessary, completed by minimally invasive percutaneous or limited open instrumentation. Kirschner wires effectively splint the reduction until fracture callus is visualised on X-ray. An occasional mini plate is required to buttress a displaced volar medial lunate facet (die punch) fragment into position. First, major metaphyseal articular fragments are approximated to restore the articular surface. Smaller fragments follow their larger counterparts into position or may be ignored (the "Rule of the Majority" or "Vassal Rule"). The repaired metaphysis is then aligned with and stabilised to the diaphysis. Cancellous bone may be inserted through small targeted incisions when defects and areas of comminution are present. The wrist is splinted in a functional (slightly extended) position for three to four weeks in uncomplicated cases. Digital elevation and rehabilitation are emphasised during the early stages of fracture healing. After callus appears on X-ray, progressive wrist rehabilitation is initiated and the patients are weaned from their splints. Minimally invasive surgical intervention, good pain control and early rehabilitation maximise functional recovery and minimise morbidity, medical costs and lost work time.

Concentration of bisphosphonate (incadronate) in callus area and its effects on fracture healing in rats.

The aim of the present study was to investigate effects of incadronate on early stages of fracture healing and to detect its concentration in callus area (Ca.Ar). Rats were injected three times per week with either two doses of incadronate (10 microg/kg and 100 microg/kg) or vehicle for 2 weeks. Femora were then fractured and fixed and animals were divided into pretreatment (P-10 and P-100) and continuous treatment (C-10 and C-100) groups. Incadronate treatment was stopped in P-10 and P-100 groups but continued in C-10 and C-100 groups. Animals were killed at 2 weeks and 4 weeks after fracture. Results showed significantly large callus, compared with the control, only in C-100 group at 4 weeks but not at 2 weeks. Both linear labeled surface (LS) and eroded surface (ES) decreased significantly in C-10 and C-100 groups at 2 weeks and 4 weeks. Osteoclast number (N.Oc) decreased significantly in C-10 and C-100 groups at 2 weeks but increased slightly at 4 weeks. However, there was no significant difference in the above parameters in P-10 and P-100 groups at 4 weeks. Apoptotic osteoclasts were observed only in the C-100 group at 4 weeks. A time-course decrease in incadronate concentration was detected in P-10 and P-100 groups whereas an increase was observed in C-10 and C-100 groups. These findings suggest that larger callus under incadronate treatment may result from the inhibition of bone resorption, histological characteristics of callus may be correlated with incadronate concentration, and metabolism of incadronate in bone may be related to the rate of bone turnover.

Is human fracture hematoma inherently angiogenic?

This study attempts to explain the cellular events characterizing the changes seen in the medullary callus adjacent to the interfragmentary hematoma during the early stages of fracture healing. It also shows that human fracture hematoma contains the angiogenic cytokine vascular endothelial growth factor and has the inherent capability to induce angiogenesis and thus promote revascularization during bone repair. Patients undergoing emergency surgery for isolated bony injury were studied. Raised circulating levels of vascular endothelial growth factor were seen in all injured patients, whereas the fracture hematoma contained significantly higher levels of vascular endothelial growth factor than did plasma from these injured patients. However, incubation of endothelial cells in fracture hematoma supernatant significantly inhibited the in vitro angiogenic parameters of endothelial cell proliferation and microtubule formation. These phenomena are dependent on a local biochemical milieu that does not support cytokinesis. The hematoma potassium concentration is cytotoxic to endothelial cells and osteoblasts. Subcutaneous transplantation of the fracture hematoma into a murine wound model resulted in new blood vessel formation after hematoma resorption. This angiogenic effect is mediated by the significant concentrations of vascular endothelial growth factor found in the hematoma. This study identifies an angiogenic cytokine involved in human fracture healing and shows that fracture hematoma is inherently angiogenic. The differences between the in vitro and in vivo findings may explain the phenomenon of interfragmentary hematoma organization and resorption that precedes fracture revascularization.