Proteasome inhibition-enhanced fracture repair is associated with increased mesenchymal progenitor cells in mice.

The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.

[Methylenetetrahydrofolate reductase polymorphism C677T in patients with consolidated fractures and pseudarthrosis of long bones: relationship with homocystein and inflammatory mediators].

In article described research the results of the prevalence of the genetic polymorphism of the gene Methylentetrahydrofolatereductase C677T (MTHFR) in 130 patients with pseudarthrosis of long bones and in those with consolidated fractures. The incidence of allele-T among patients with pseudarthrosis was 1.4 times higher than among those with consolidated fractures. Pathological genotype MTHFR 677-TT was associated with the development avital types of pseudarthrosis and increase the proportion of people with hyperhomocysteinemia, high content of inflammatory mediators and development refracture.

The effect of Cox-2 specific inhibition on direct fracture healing in the rabbit tibia.

OBJECTIVE: The purpose of this study was to evaluate the effect of Cox-2 administration on direct (primary) fracture healing. METHODS: A transverse tibial osteotomy was created in adult male rabbits and rigidly fixed in compression using a 2.7-mm dynamic compression plate. Animals were randomized to receive either rofecoxib (12.5 mg orally per day) or placebo. Animals were killed at 4 weeks and fracture healing assessed by mechanical testing. RESULTS: There were no significant differences between the control and Cox-2 treated animals in terms of mechanical strength at 4 weeks. There was a high complication rate of peri-implant fractures during the daily medication administration. CONCLUSION: The immediate administration of a Cox-2 specific inhibitor did not impair primary (direct) bone healing at the dose administered in this rabbit tibial osteotomy model.

Low-level laser therapy modulates cyclo-oxygenase-2 expression during bone repair in rats.

The goal of this study was to analyze the role of cyclo-oxygenase-2 following bone repair in rats submitted to low-level laser therapy. A total of 48 rats underwent surgery to inflict bone defects in their tibias having been randomly distributed into two groups: negative control and laser exposed group, i.e., the animals were treated with low-level laser therapy by means of gallium arsenide laser at 16 J/cm(2). The animals were killed after 48 h, 7 days, 14 days, or 21 days. The tibias were removed for morphological, morphometric, and immunohistochemistry analysis for cyclo-oxygenase-2. Statistical significant differences (P < 0.05) were observed in the quality of bone repair and quantity of formed bone between groups 14 days after surgery in the laser exposed group. In the same way, cyclo-oxygenase-2 immunoreactivity was more intense in bone cells for intermediate periods evaluated in this group. Taken together, such results suggest that low-level laser therapy is able to improve bone repair in the tibia of rats after 14 days of surgery as a result of an up-regulation for cyclo-oxygenase-2 expression in bone cells.

Effects of imiglucerase treatment on traumatic fracture and bone and blood abnormalities in a patient with previously untreated type 1 gaucher disease.

OBJECTIVE: This letter reports on the effect of enzyme replacement therapy with imiglucerase on bone healing and bone and blood abnormalities in a woman with previously untreated type 1 Gaucher disease (GD). METHODS: The 49-year-old patient had been diagnosed with GD at the age of 28 years and had previously undergone splenectomy. She presented with pseudarthrosis 14 months after sustaining a traumatic fracture of the tibia and fibula. Therapy was begun with imiglucerase 60 U/kg q2wk. The effects of treatment on bone healing were monitored radiographically, and effects on blood and bone marrow biology were monitored by hemograms, myelograms, and hematopoietic and mesenchymal clonogenic assays. RESULTS: Objective bone healing was observed starting in the third month of imiglucerase treatment. Blood abnormalities normalized and bone marrow parameters improved over the first 9 months, including a decrease in Gaucher cells, an increase in bone marrow CD34+ cell cloning efficiency, and the appearance of mesenchymal progenitors. CONCLUSION: This research letter reports the results of hematologic and bone evaluations during enzyme replacement therapy with imiglucerase in a woman with previously untreated type 1 GD who presented with a traumatic fracture.

Complex tibial fracture outcomes following treatment with low-intensity pulsed ultrasound.

A clinical study was conducted to investigate the effect of low-intensity pulsed ultrasound (US) stimulation (LIPUS) on the healing of complex tibial fractures. Thirty complex tibial fractures were randomly assigned to the treatment with LIPUS (n = 16) or by a dummy machine (sham-exposed: n = 14). The fractures were immobilized by either internal or external fixations according to the clinical indications. LIPUS was given 20 min/day for 90 days. Fracture healing was monitored by clinical, radiological, densitometric and biochemical assessments. The LIPUS-treated group showed statistically significantly better healing, as demonstrated by all assessments. Complications were minimal in the LIPUS group. There were two cases of delayed union, with one in each group. There were two cases of infection in the control group. The delayed-union cases were subsequently treated by LIPUS and the infection cases were treated with standard protocol. Fracture healing in these patients was again treated by LIPUS.

Heparanase mRNA expression during fracture repair in mice.

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.

Matrix metalloproteinase-2 and -9 are activated in joint diseases.

A study was performed to identify the activation status of the gelatinase MMPs, MMP-2 and -9, in both normal and diseased equine articular tissues. In addition, the production and activation status of equine MMP-2 and -9 by equine articular cells and tissues in response to increasing IL-1beta concentrations was assessed. The study was performed to test the hypothesis that activation of MMPs is a fundamental step in the pathogenesis of joint diseases; and that this activation is mediated by the cytokine IL-1. Using purified equine MMP-2 and -9, the molecular weights of the zymogen and activated form of equine MMP-2 and -9 were identified by a combination of gelatin zymography and a gelatin degradation assay using aminophenylmercuric acetate as a chemical activator of the molecules. Normal equine articular tissues (cartilage and synovial membrane) maintained in short-term tissue culture produced MMP-2 zymogen alone, while similar tissues obtained from a variety of pathological conditions produce both zymogen and active MMP-2, as well as MMP-9 monomer and dimer. Activated MMP-9 was an inconsistent finding. Normal equine synovial fibroblasts in monolayer culture produced zymogen MMP-2 alone under basal conditions. A mild increase in active and zymogen MMP-2 levels occurred with IL-1beta treatment. Equine synovial membrane explants demonstrated a dose-dependent increase in active and zymogen MMP-2 and MMP-9 levels following IL-1beta treatment. Monolayer chondrocyte cell cultures demonstrated a dose-dependent mild increase in active and zymogen MMP-2 following IL-1beta treatment. Explant cartilage cultures demonstrated a dose-dependent mild increase in zymogen MMP-2 alone following IL-1beta treatment. This study supports the hypothesis that activation of MMPs is occurring in joint disease, and that in vitro stimulation of equine articular cells and tissues causes not only an increase in MMP production, but also an increase in amount of activated enzyme released. Further research is required to investigate the role of MMP activation in joint diseases, and to investigate the potential use of therapeutic agents, which inhibit MMP activation, in the treatment and prevention of joint diseases.

Is acid phosphatase activity present in bone matrix at sites of endochondral ossification in rabbit fracture callus?

It has been suggested that acid phosphatase activity is present in newly formed bone matrix at sites of endochondral ossification in rabbit fracture calluses. Because acid phosphatases are usually found intracellularly, it was decided to test this possibility more rigorously. Tissue from 10- and 14-day healing rabbit fractures was subjected to a series of critical tests for acid phosphatases with a pH optimum of 5.0. Fluoride, tartrate and molybdate were used as potential inhibitors of acid phosphatase activity. The effects of several counterstaining protocols were also investigated. A fluoride- and tartrate-resistant acid phosphatase is located in osteoclasts and mononuclear phagocytes. Diffuse staining of the bone matrix is seen, but it is dependent upon the length of incubation in the substrate medium and the distance from the acid phosphatase-reacting cells. It is concluded that the coloration of the bone matrix is probably caused by diffusion of the dye and reaction product and is, therefore, artifactual.

Three-fold induction of renal 25-hydroxyvitamin D3-24-hydroxylase activity and increased serum 24,25-dihydroxyvitamin D3 levels are correlated with the healing process after chick tibial fracture.

To investigate the possible biological actions of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), a tibial fracture-healing model was established in White Leghorn chicks. Three-week-old White Leghorn chicks fed a vitamin D3-replete diet were divided into four groups (control, anesthetized, sham, and fractured). On varying days after tibial fracture (F) or sham manipulation (S), renal 25(OH)D3-1 alpha-hydroxylase and 25(OH)D3-24-hydroxylase (24-hydroxylase) activities and serum Ca2+ concentrations were measured. Metofane anesthesia was found to have no effect on the activity of either of the hydroxylases; the activities of the hydroxylases in the control, anesthetized, and sham-operated birds were similar. By 10 days after tibial fracture, the renal 24-hydroxylase activity increased more than 3-fold in F (1.33 +/- 0.07 pmol/mg of protein) as compared with S (0.42 +/- 0.03 pmol/mg of protein) (p < 0.0001). A time-dependent study of the renal 24-hydroxylase activity during the fracture repair process revealed a slow increase from the first day after fracture, a higher activity at 8 days, which peaked at 10-11 days, which is consistent with the formation of the callus. The 24-hydroxylase activity then returned to the same level as the sham group 14 days after fracture. There was no significant difference in serum Ca2+ levels between the F and S groups over the 3-week postfracture period. Serum levels of vitamin D3 metabolites were also measured during the fracture healing process: a 3.4x increase of the 24,25(OH)2D3 level in the fractured group (3.64 +/- 1.16 nM) was observed as compared with the control groups (1.08 +/- 0.49 nM) at 10 days after fracture (p = 0.068). No significant differences were observed in the plasma levels of 25(OH)D3 or 1 alpha, 25(OH)2D3 between the group with a fracture and the controls. Exposure of primary chick kidney cells in culture to serum obtained from chicks with a tibial fracture for 20 h resulted in an approximately 40% increase in the activity of the 24-hydroxylase as compared with cells exposed to serum from control birds. These results suggest that 24,25(OH)2D3 is involved in the early process of fracture repair and that there is some form of physiological communication between the fractured bone and the kidney so as to increase the renal 24-hydroxylase and the circulating concentration of this metabolite.

Group II phospholipase A2 in serum after knee surgery and intramedullary nailing of tibial shaft fracture.

This prospective study investigates the effect of injury and surgery of cartilage and bone on serum group II phospholipase A2 (PLA2-II) and C-reactive protein (CRP) levels. Serum concentrations of PLA2-II and CRP were measured before and after the operation in nine patients with closed tibial shaft fractures treated by nailing, 11 patients with fractures of lateral tibial plateau treated by bone grafting, and 19 patients with ruptured anterior cruciate ligament treated by reconstruction. The postoperative PLA2-II and CRP values were statistically significantly higher than the pre-operative values in the tibial plateau fracture and ligament rupture groups, whereas the increase in the PLA2-II values in the tibial fracture group was not statistically significant. The highest values of both parameters were found on the second postoperative day. The changes in the PLA2-II and CRP values were parallel in the lateral condyle fracture and in anterior cruciate ligament rupture groups. PLA2-II behaves as an acute phase reactant in the serum of patients undergoing acute and elective knee surgery.

Serum osteocalcin and total alkaline phosphatase levels as prognostic indicators in tibial shaft fractures.

Serum samples were obtained periodically from 50 adult patients with closed tibial shaft fractures. Total alkaline phosphatase activity was measured in all cases and osteocalcin activity was measured in 14 patients. Fractures produced by high-energy violence generally had lower values of osteocalcin activity. This could be due to depressed circulating vitamin K levels throughout the healing period in the former fractures. In addition, normally uniting fractures had generally higher values of osteocalcin activity compared with fractures exhibiting delayed union. This indicates depressed osteoblastic activity in slowly healing fractures. The results suggest that measurement of osteocalcin activity after fracture could provide a useful prognostic indicator. By contrast, total serum alkaline phosphatase activity was not significantly different between the injury groups and between the healing groups.

[Serum results following experimental fracture alignment and thyrocalcitonin treatment (author's transl)]. / Klinisch-chemische Serumbefunde nach experimenteller Fraktursetzung und Calcitonin-Behandlung.

Serum alkaline phosphatase activity, serum calcium and phosphorus concentrations were determined during bone healing under treatment of thyrocalcitonin. The right tibiae of 160 albino rats were osteotomized. Of these, 80 were treated with thyrocalcitonin. During the 50-day observation period, the calcium level in the thyrocalcitonin-treated sample never differed significantly from that of the control group. However, the alkaline phosphatase activity and the serum phosphorus level significantly differed from the control levels (P less than 0.05) during 4 days of observation. The increased serum alkaline phoshatase activity--a sign of osteoblastic activity--suggests that thyrocalcitonin may aid in osteogenesis and bone healing.

Creatine kinase in tibial shaft fractures. Serum creatine kinase levels in patients with tibial shaft fractures.

The creatine kinase (CK) activity of the serum of 33 male and 24 female patients with tibial shaft fractures has been assayed. In 40 of the 57 patients the CK level surpassed the maximal normal limit of 1.7 mukat/l. Patients with fractures due to direct violence had significantly higher levels than those with fractures due to indirect violence. When the fracture was displaced the CK level was more often abnormal than when there was no displacement. Patients with extensive swelling of the injured leg had significantly higher levels than patients with minor or no swelling. CK determinations could be used to quantify muscle injury.

[Alkaline phosphatase of granulocytes in open fractures of the long tubular bones]. / Shchelochnaia fosfataza granulotsitov pri otkrytykh perelomakh dlinnykh trubchatykh kostei

Investigations carried out in 60 patients with open fractures of long tubular bones in osteosynthesis with the Ilizarov apparatus indicated that alkaline phosphatase (AP) of granulocytes fully reflects the postoperative course. 10--24 hours following the trauma the level of AP in patients with grave injuries exceeds that in patients with slight ones. The differences are found to be reliable from the 1st to the 5th day after the injury.

Leukocyte and serum alkaline phosphatase after hip arthroplasty, synovectomy of the knee and fracture.

The alkaline phosphatase in leukocytes (LAP) was measured in patients who had undergone various types of total arthroplasty of the hip, in patients who had undergone synovectomy and in whom tibial fractures had been treated conservatively. The LAP-Score increased rapidly and reached a maximum within a few days and returned to original levels after 14 days both after soft tissue trauma and skeletal trauma. The size of the maximal increase appears to vary only with the severity of the trauma. LAP was not found to be correlated with the increase in alkaline phosphatase in the serum. The increase in serum alkaline phosphatase occurred much later and decreased much slower and occurred only after skeletal trauma. The increase in alkaline phosphatase in the serum appears to vary with severity of skeletal trauma. A significant difference in LAP (P less than 0.05) was found between patients operated upon with total arthroplasty with success and those with deep infection. The difference between these two groups was, however, larger (P less than 0.01) regarding ESR and CRP.