Richard M. Terek

Defects of early fracture-healing in experimental diabetes.

Diabetes has been implicated as a cause of impaired fracture-healing. To test this hypothesis, we tested the tensile strength of femora from normal rats and from untreated and insulin-treated diabetic rats two weeks after the production of a closed fracture. One week before the fracture, diabetes was induced by administration of streptozotocin (sixty-five milligrams per kilogram of body weight). The concentration of serum glucose increased from 6.1 +/- 0.3 millimoles per liter (110 +/- 5 milligrams per deciliter) in the control animals to 31.1 +/- 0.8 millimoles per liter (560 +/- 15 milligrams per deciliter) in the untreated diabetic animals. After two weeks of healing, fracture callus from the untreated diabetic animals had a 29 per cent decrease in tensile strength and a 50 per cent decrease in stiffness compared with the controls. Treatment of the diabetic animals with insulin resulted in a mean concentration of serum glucose of 14.4 +/- 0.6 millimoles per liter (260 +/- 10 milligrams per deciliter) and restored the tensile strength and stiffness of the callus to a value that was not statistically different from that of the controls. Between the fourth and eleventh days of healing, there was a 50 to 55 per cent decrease in the collagen content of the callus of the untreated diabetic animals compared with the controls. In addition, on the fourth day of healing, DNA content, an indicator of cellularity of the callus, was decreased 40 per cent in the untreated diabetic group. Between the fourth and eleventh days of healing, the collagen-to-DNA ratio, which was determined as an indicator of net collagen synthesis per cell, was decreased 15 to 50 per cent in callus from the untreated diabetic animals.

Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling.

The tyrosine phosphatase SHP2, encoded by PTPN11, is required for the survival, proliferation and differentiation of various cell types. Germline activating mutations in PTPN11 cause Noonan syndrome, whereas somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumours. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, enchondromas, joint destruction and bony deformities. The detailed pathogenesis of this disorder has remained unclear. Here we use a conditional knockout (floxed) Ptpn11 allele (Ptpn11fl) and Cre recombinase transgenic mice to delete Ptpn11 specifically in monocytes, macrophages and osteoclasts (lysozyme M-Cre; LysMCre) or in cathepsin K (Ctsk)-expressing cells, previously thought to be osteoclasts. LysMCre;Ptpn11fl/fl mice had mild osteopetrosis. Notably, however, CtskCre;Ptpn11fl/fl mice developed features very similar to metachondromatosis. Lineage tracing revealed a novel population of CtskCre-expressing cells in the perichondrial groove of Ranvier that display markers and functional properties consistent with mesenchymal progenitors. Chondroid neoplasms arise from these cells and show decreased extracellular signal-regulated kinase (ERK) pathway activation, increased Indian hedgehog (Ihh) and parathyroid hormone-related protein (Pthrp, also known as Pthlh) expression and excessive proliferation. Shp2-deficient chondroprogenitors had decreased fibroblast growth factor-evoked ERK activation and enhanced Ihh and Pthrp expression, whereas fibroblast growth factor receptor (FGFR) or mitogen-activated protein kinase kinase (MEK) inhibitor treatment of chondroid cells increased Ihh and Pthrp expression. Importantly, smoothened inhibitor treatment ameliorated metachondromatosis features in CtskCre;Ptpn11fl/fl mice. Thus, in contrast to its pro-oncogenic role in haematopoietic and epithelial cells, Ptpn11 is a tumour suppressor in cartilage, acting through a FGFR/MEK/ERK-dependent pathway in a novel progenitor cell population to prevent excessive Ihh production.

Treatment of soft-tissue sarcomas of the hand.

We studied the clinical features, radiographic and pathological findings, treatment, and results for twenty-three patients who had been managed for a soft-tissue sarcoma of the hand between 1982 and 1990. The ages of the patients ranged from sixteen to seventy-six years (median age, thirty-one years). The most common clinical finding was a small, painless soft-tissue mass. Twenty of the tumors were high-grade, and eighteen were less than five centimeters in diameter. The most common diagnosis was synovial sarcoma, which was identified in eight patients. Leiomyosarcoma, rhabdomyosarcoma, and malignant fibrous histiocytoma developed in three patients each; epithelioid sarcoma, in two patients; and angiosarcoma, liposarcoma, neuroectodermal tumor, and clear-cell sarcoma, in one patient each. Curative wide excision or amputation was attempted in twenty-two patients; the margins were positive for tumor cells in eight, and local recurrence was seen in nine. Of the twenty-three patients, fourteen had survived, without evidence of disease, after a median duration of follow-up of forty-nine months, and nine had died of disease. The median rate of survival did not differ significantly on the basis of the size or grade of the tumor or the use of adjuvant treatment. However, the rate of survival of the patients who had a soft-tissue sarcoma of the hand that was less than five centimeters in diameter was significantly lower (p = 0.0008) than that of 152 patients who had a similar tumor at another site in an extremity.(ABSTRACT TRUNCATED AT 250 WORDS)

Allograft reconstruction after proximal tibial resection for bone tumors. An analysis of function and outcome comparing allograft and prosthetic reconstructions.

Seventeen patients (age, 12-63 years; median, 22 years) treated with proximal tibial allografts were identified. Nine cases were intercalary and eight were osteoarticular allografts. Complications, number of operations, and oncologic and functional results were reviewed. The functional results of the allografts were compared with a prior cohort of patients who had endoprosthesis at the same institution by the same surgeons. There were 14 malignant tumors, two benign aggressive tumors, and one sclerosing osteomyelitis mimicking osteosarcoma. Twelve of 17 patients had complications, the most common being fracture, deformity, and infection. Six patients required more than one procedure, and three had amputations after allograft reconstruction. The ultimate function was excellent in three patients, good in seven, fair in six, and poor in one. There were 14 patients with endoprosthetic reconstruction. Wound problems followed by prosthetic loosening were the most common complications. Of the eight patients requiring a second procedure, three had an amputation. Three had excellent, seven good, and four fair functional results at the final evaluation. No patient in either group had a local recurrence. Allograft provides an alternative to endoprosthetic reconstruction; however, the high incidence of complications makes the outcome unpredictable. Allograft or prosthetic reconstruction provides better functional results than amputation without sacrificing oncologic results.

Activation of Indian hedgehog promotes chondrocyte hypertrophy and upregulation of MMP-13 in human osteoarthritic cartilage

OBJECTIVE The objectives of this study were to (1) determine the correlation between osteoarthritis (OA) and Indian hedgehog (Ihh) expression, and (2) establish the effects of Ihh on expression of markers of chondrocyte hypertrophy and matrix metalloprotease (MMP)-13 in human OA cartilage. DESIGN OA cartilage and synovial fluid samples were obtained during total knee arthroplasty. Normal cartilage samples were obtained from intra-articular tumor resections, and normal synovial fluid samples were obtained from healthy volunteers and the contralateral uninjured knee of patients undergoing anterior cruciate ligament reconstruction. OA was graded using the Mankin score. Expression of Ihh in synovial fluid was determined by Western blot. Ihh, type X collagen and MMP-13 mRNA were determined by real time PCR. Protein expression of type X collagen and MMP-13 in cartilage samples was analyzed with immunohistochemistry. Chondrocyte size was measured using image analysis. RESULTS Ihh expression was increased 2.6 fold in OA cartilage and 37% in OA synovial fluid when compared to normal control samples. Increased expression of Ihh was associated with the severity of OA and expression of markers of chondrocyte hypertrophy: type X collagen and MMP-13, and chondocyte size. Chondrocytes were more spherical with increasing severity of OA. There was a significant correlation between Mankin score and cell size (r(2) = 0.80) and Ihh intensity (r(2) = 0.89). Exogenous Ihh induced a 6.8 fold increase of type X collagen and 2.8 fold increase of MMP-13 mRNA expression in cultured chondrocytes. Conversely, knockdown of Ihh by siRNA and Hh inhibitor cyclopamine had the opposite effect. CONCLUSIONS Ihh expression correlates with OA progression and changes in chondrocyte morphology and gene expression consistent with chondrocyte hypertrophy and cartilage degradation seen in OA cartilage. Thus, Ihh may be a potential therapeutic target to prevent OA progression.

CXCR4/SDF1 mediate hypoxia induced chondrosarcoma cell invasion through ERK signaling and increased MMP1 expression

BackgroundChondrosarcoma is a disease that does not respond to conventional cytotoxic chemotherapy and expression of MMP1 is a marker for a poor prognosis. The mechanism of increased MMP1 expression in chondrosarcoma is not completely known. Our goal is to identify molecular pathways that could serve as therapeutic targets. Chondrosarcoma become hypoxic as they grow, are capable of eliciting an angiogenic response, and typically metastasize to the lungs. The present study determined the effect of hypoxia and specifically HIF-1a on expression of CXCR4 and MMP1 and their role in chondrosarcoma cell invasion.ResultsCXCR4 and its ligand, SDF1, are upregulated in primary chondrosarcoma tumors compared to normal articular cartilage, and CXCR4 was upregulated in chondrosarcoma cell line JJ compared to normal chondrocytes. Hypoxia and specifically HIF-1a increased CXCR4 and MMP1 expression in JJ cell line and chondrosarcoma invasion in vitro. The hypoxia mediated increase in MMP1 expression and chondrosarcoma invasion could be inhibited by siRNA directed at HIF-1a or CXCR4, the CXCR4 inhibitor AMD3100, as well as with ERK inhibitor U0126 and ERK siRNA.ConclusionsChondrosarcoma cell invasion is increased by hypoxia induced expression of CXCR4 and MMP1 and is mediated by HIF-1a and ERK. Both invasion and MMP1 can be inhibited with CXCR4 blockade, suggesting that CXCR4/SDF1 signaling may be a therapeutic target for chondrosarcoma.

Stimulation of chondrocyte hypertrophy by chemokine stromal cell-derived factor 1 in the chondro-osseous junction during endochondral bone formation

During endochondral bone formation, chondrocytes undergo differentiation toward hypertrophy before they are replaced by bone and bone marrow. In this study, we found that a G-protein coupled receptor CXCR4 is predominantly expressed in hypertrophic chondrocytes, while its ligand, chemokine stromal cell-derived factor 1 (SDF-1) is expressed in the bone marrow adjacent to hypertrophic chondrocytes. Thus, they are expressed in a complementary pattern in the chondro-osseous junction of the growth plate. Transfection of a CXCR4 cDNA into pre-hypertrophic chondrocytes results in a dose-dependent increase of hypertrophic markers including Runx2, Col X, and MMP-13 in response to SDF-1 treatment. In organ culture SDF-1 infiltrates cartilage and accelerates growth plate hypertrophy. Furthermore, a continuous infusion of SDF-1 into the rabbit proximal tibial physis results in early physeal closure, which is accompanied by a transient elevation of type X collagen expression. Blocking SDF-1/CXCR4 interaction suppresses the expression of Runx2. Thus, interaction of SDF-1 and CXCR4 is required for Runx2 expression. Interestingly, knocking down Runx2 gene expression results in a decrease of CXCR4 mRNA levels in hypertrophic chondrocytes. This suggests a positive feedback loop of stimulation of chondrocyte hypertrophy by SDF-1/CXCR4, which is mediated by Runx2.

HDAC4 Represses Vascular Endothelial Growth Factor Expression in Chondrosarcoma by Modulating RUNX2 Activity

Chondrosarcoma is a primary bone tumor with a dismal prognosis; most patients with this disease develop fatal pulmonary metastases, suggesting the need for a better systemic treatment. Anti-angiogenesis treatment may be useful, because angiogenesis is critical for both tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is the most potent pro-angiogenic factor and is regulated by pathways related to the normal physiologic response to hypoxia and genetic alterations related to the malignant phenotype. Our prior work has shown that VEGF is overexpressed in high grade chondrosarcoma and chondrosarcoma cell lines. Working on the premise that developmental pathways giving a selective growth advantage are often recapitulated in tumors, we investigated the regulation of VEGF by HDAC4 and Runx2 in chondrosarcoma. We tested the hypothesis that there is dysregulation of HDAC4/Runx2/VEGF gene expression and that decreased HDAC4 expression accounts for at least some of the increased VEGF expression seen in chondrosarcoma. We show that reduced expression of HDAC4 in chondrosarcoma cells increases expression of Runx2 leading to increased expression of VEGF and in vitro angiogenesis. Thus, both hypoxia and dysregulated expression of a developmental pathway are causes of increased VEGF expression in chondrosarcoma.

Gadolinium inhibits thymidine incorporation and induces apoptosis in chondrocytes

Magnetic resonance arthrography, a procedure where contrast agents containing gadolinium are administered intra‐articularly, has become a useful tool in musculoskeletal diagnosis. Although considered safe for systemic use, toxicities in some tissues have been identified for both free gadolinium ion and the gadolinium chelates used as contrast. In this study, the effects of short‐term exposure of articular chondrocytes to gadolinium contrast were examined by assaying for proteoglycan synthesis, cell proliferation, and apoptosis. Bovine chondrocytes were grown in monolayer culture and exposed to gadodiamide for 16 h. Proteoglycan synthesis was measured through incorporation of radiolabeled sulfate. Uptake of radiolabeled thymidine assessed cell proliferation. Apoptosis was detected using the TUNEL assay, where DNA strand breaks characteristic of apoptosis are labeled with fluorescent nucleotide. Proteoglycan synthesis was stimulated by lower dose exposure to gadodiamide. At higher doses, proteoglycan synthesis returned to baseline. Cell proliferation decreased following exposure to gadodiamide in a dose‐dependent manner. Chondrocyte apoptosis was induced in a dose‐dependent manner. Further work is needed to determine if these in vitro effects are present in the intact joint.

The expression of platelet-derived growth-factor gene in Dupuytren contracture.

Dupuytren contracture is a disease of the palmar fascia characterized by nodular fibroblastic proliferation; its etiology and pathogenesis are poorly understood. Growth factors are polypeptides that regulate cell growth and differentiation and extracellular matrix production. Platelet-derived growth factor is known to cause fibroblastic proliferation, and it may be involved in the pathogenesis of Dupuytren contracture. The purpose of this study was to determine if the gene for the B chain of platelet-derived growth factor is expressed in Dupuytren contracture. Tissue from patients who had Dupuytren disease was examined immunohistochemically with the 5B5 antibody, which is a marker for fibroblasts. Polymerase chain reaction, gel electrophoresis, Southern blotting, and in situ hybridization were also used to study gene expression in the tissue as well as in normal fascia, A172 cells, and MRC5 cells. Total cellular RNA was extracted from tissue and cells. Polymerase chain reaction was done with oligonucleotide primers complementary to a portion of the platelet-derived growth-factor-B and platelet-derived growth-factor-receptor genes. The platelet-derived growth-factor-B gene was expressed in all six specimens from the patients who had Dupuytren contracture as well as in the A172 cells, but not in the normal fascia lata or the MRC5 cells. These results were confirmed with Southern blotting of the products of the reaction with a platelet-derived growth-factor-B probe. The gene for the platelet-derived growth-factor receptor was expressed by all tissues and cells studied.(ABSTRACT TRUNCATED AT 250 WORDS)