Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

The use of routine thoracoabdominal CT scans in the polytrauma patient to estimate obesity.

OBJECTIVE: To utilize data from routine CT scans to quantify obesity in polytrauma patients without the need to obtain a height and weight. DESIGN AND METHODS: We utilized a comprehensive database including multidetector CT thoracoabdominal images of all polytrauma patients admitted to a Level 1 trauma center. One thousand one hundred seventy-four patients were reviewed from 2006 to 2008 and of these, 162 had previous documentation of Body Mass Index (BMI) or height and weight measurements as an outpatient within 6 months of trauma activation and with a truncal girth smaller than the scanning area of the CT machine. Truncal Adiposity Volume (TAV) was calculated from three dimensional reconstructions (3DRs) of the CT scans of the thorax and abdomen obtained in the emergency department. RESULTS: Statistical analysis yielded a fairly good correlation between TAV and BMI (correlation coefficient = 0.77; p-value < 0.0001). The intra-observer and inter-observer correlations in measuring TAV were high; 0.99 and 0.98 respectively. A linear regression equation of BMI on TAV was estimated and it had a form: 3DR BMI = 20.81+0.00064×TAV. In conclusion, TAV provides a reproducible means of evaluating obesity in trauma patients from routinely obtained CT scans. CONCLUSIONS: The TAV eliminates the often problematic task of obtaining a height and weight in a trauma patient and it correlates fairly well with the most commonly used clinical method of quantifying patient adiposity, BMI. This method may provide a more direct measurement of adiposity than does BMI, and holds promise for improving trauma care and research in the obese patient.

Marrow stimulation improves meniscal healing at early endpoints in a rabbit meniscal injury model.

PURPOSE: To critically evaluate the effect of marrow stimulation (MS) on the extent of healing and the local biological environment after meniscal injury in ligamentously stable knees in a rabbit model. METHODS: A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in 18 New Zealand White rabbits (36 knees). In right knees (MS knees), a 2.4-mm Steinman pin was drilled into the apex of the femoral intercondylar notch and marrow contents were observed spilling into the joint. Left knees served as controls. Rabbits were killed in 3 groups (n = 6 rabbits each) at 1, 4, and 12 weeks with meniscal harvest and blinded histomorphometric and histologic evaluation using an established 3-component tissue quality score (range, 0 to 6). One-week specimens were also evaluated for the presence of proregenerative cytokines using immunohistochemistry. RESULTS: The mean proportion of the avascular zone defect bridged by reparative tissue was greater in MS knees than in controls at each endpoint (1 week, 55% v 30%, P = .02; 4 weeks, 71% v 53%, P = .047; 12 weeks, 96% v 77%, P = .16). Similarly, there was a consistent trend toward superior tissue quality scores in knees treated with MS compared with controls (1 week, 1.8 v 0.3, P = .03; 4 weeks, 4.3 v 2.8, P = .08; 12 weeks, 5.9 v 4.5, P = .21). No statistically significant differences, however, were observed at the 12-week endpoint. Increased staining for insulin-like growth factor I, transforming growth factor-ß, and platelet-derived growth factor was observed in regenerated tissue, compared with native meniscal tissue, in all specimens at 1 week. Staining density for all growth factors was similar, however, in reparative tissue of MS and control knees. CONCLUSIONS: The results of this study suggest that marrow stimulation leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period. CLINICAL RELEVANCE: Clinical evaluation of marrow stimulation techniques designed to enhance healing in isolated meniscus repair surgery may be indicated.

Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration.

The methodology for the repair of critical-sized or non-union bone lesions has unpredictable efficacy due in part to our incomplete knowledge of bone repair and the biocompatibility of bone substitutes. Although human mesenchymal stem cells (hMSCs) differentiate into osteoblasts, which promote bone growth, their ability to repair bone in vivo has been variable. We hypothesized that given the multistage process of osteogenesis, hMSC-mediated repair might be maximal at a specific time point of healing. Using a mouse model of calvarial healing, we demonstrate that the osteo-repair capacity of hMSCs can be substantially augmented by treatment with an inhibitor of peroxisome proliferator-activated receptor γ, but efficacy is confined to the rapid osteogenic phase. Upon entry into the bone-remodeling phase, hMSC retention signals are lost, resulting in truncation of healing. To solve this limitation, we prepared a scaffold consisting of hMSC-derived extracellular matrix (ECM) containing the necessary biomolecules for extended site-specific hMSC retention. When inhibitor-treated hMSCs were coadministered with ECM, they remained at the injury, well into the remodeling phase of healing, which resulted in reproducible and complete repair of critical-sized bone defects in mice in 3 weeks. These data suggest that hMSC-derived ECM and inhibitor-treated hMSCs could be used at optimal times to substantially and reproducibly improve bone repair.

Implantation of allogenic synovial stem cells promotes meniscal regeneration in a rabbit meniscal defect model.

BACKGROUND: Indications for surgical meniscal repair are limited, and failure rates remain high. Thus, new ways to augment repair and stimulate meniscal regeneration are needed. Mesenchymal stem cells are multipotent cells present in mature individuals and accessible from peripheral connective tissue sites, including synovium. The purpose of this study was to quantitatively evaluate the effect of implantation of synovial tissue-derived mesenchymal stem cells on meniscal regeneration in a rabbit model of partial meniscectomy. METHODS: Synovial mesenchymal stem cells were harvested from the knee of one New Zealand White rabbit, expanded in culture, and labeled with a fluorescent marker. A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in fifteen additional rabbits. Allogenic synovial mesenchymal stem cells suspended in phosphate-buffered saline solution were implanted into the right knees, and phosphate-buffered saline solution alone was placed in the left knees. Meniscal regeneration was evaluated histologically at four, twelve, and twenty-four weeks for (1) quantity and (2) quality (with use of an established three-component scoring system). A similar procedure was performed in four additional rabbits with use of green fluorescent protein-positive synovial mesenchymal stem cells for the purpose of tracking progeny following implantation. RESULTS: The quantity of regenerated tissue in the group that had implantation of synovial mesenchymal stem cells was greater at all end points, reaching significance at four and twelve weeks (p < 0.05). Tissue quality scores were also superior in knees treated with mesenchymal stem cells compared with controls at all end points, achieving significance at twelve and twenty-four weeks (3.8 versus 2.8 at four weeks [p = 0.29], 5.7 versus 1.7 at twelve weeks [p = 0.008], and 6.0 versus 3.9 at twenty-four weeks [p = 0.021]). Implanted cells adhered to meniscal defects and were observed in the regenerated tissue, where they differentiated into type-I and II collagen-expressing cells, at up to twenty-four weeks. CONCLUSIONS: Synovial mesenchymal stem cells adhere to sites of meniscal injury, differentiate into cells resembling meniscal fibrochondrocytes, and enhance both quality and quantity of meniscal regeneration.

Alcohol induced epigenetic perturbations during the inflammatory stage of fracture healing.

It is well recognized by orthopedic surgeons that fractures of alcoholics are more difficult to heal successfully and have a higher incidence of non-union, but the mechanism of alcohol's effect on fracture healing is unknown. In order to give direction for the study of the effects of alcohol on fracture healing, we propose to identify gene expression and microRNA changes during the early stages of fracture healing that might be attributable to alcohol consumption. As the inflammatory stage appears to be the most critical for successful fracture healing, this paper focuses on the events at day three following fracture or the stage of inflammation. Sprague-Dawley rats were placed on an ethanol-containing or pair-fed Lieber and DeCarli diet for four weeks prior to surgical fracture. Following insertion of a medullary pin, a closed mid-diaphyseal fracture was induced using a Bonnarens and Einhorn fracture device. At three days' post-fracture, the region of the fracture calluses was harvested from the right hind-limb. RNA was extracted and microarray analysis was conducted against the entire rat genome. There were 35 genes that demonstrated significant increased expression due to alcohol consumption and 20 that decreased due to alcohol. In addition, the expression of 20 microRNAs was increased and six decreased. In summary, while it is recognized that mRNA levels may or may not represent protein levels successfully produced by the cell, these studies reveal changes in gene expression that support the hypothesis that alcohol consumption affects events involved with inflammation. MicroRNAs are known to modulate mRNA and these findings were consistent with much of what was seen with mRNA microarray analysis, especially the involvement of smad4 which was demonstrated by mRNA microarray, microRNA and polymerase chain reaction.

Trabecular quality and cellular characteristics of normal, diabetic, and charcot bone.

Charcot neuroarthropathy (CN) is a disabling and devastating condition that affects many neuropathic diabetic patients. It can lead to foot deformity, ulceration, and lower extremity amputation. The pathogenesis of CN is not clear, but 1 possible predisposing factor is increased bone turnover and increased osteoclastic activity. Although the affect of diabetes on bone is not entirely clear, studies have shown increased bone fragility in diabetics with neuropathy. The purpose of the present study was to compare the bone quality histologic findings and trabecular histomorphometry, including the cellular characteristics, between normal subjects (N = 7), diabetics without CN (N = 8), and diabetics with CN (N = 8). Histologically, the bone in diabetics with CN displayed an inflammatory, myxoid infiltrate. We also observed a statistically significant decrease in the number of trabeculae in bone in diabetics with CN compared with normal controls (p < .02). However, the difference between the trabeculae in diabetics with CN and diabetics without CN was not statistically significant (p > .05). Histologically, the CN bone appeared to be infiltrated with inflammatory myxoid tissue and had a disorganized trabecular pattern compared with diabetic bone without CN and normal bone. The trabeculae in patients with CN appeared to have poor quality characteristics compared with that of the other groups. The findings from the present study might indicate that diabetes mellitus bone is fragile, and the decrease in the cellular component might impair the reparative process in those with CN foot.

Cytokine-mediated inflammatory reaction following posterior cervical decompression and fusion associated with recombinant human bone morphogenetic protein-2: a case study.

STUDY DESIGN: Case study with unique laboratory analysis. OBJECTIVE: To present a potentially serious adverse event that may occur in unique individuals when using recombinant human bone morphogenetic protein-2 (rhBMP-2) to augment fusion in posterior cervical spine surgery. SUMMARY OF BACKGROUND DATA: The use of rhBMP-2 to augment posterior cervical decompression and fusion has not been approved by the Food and Drug Administration but has been advocated as safe to use by case series studies and multiple authors. METHODS: A 66-year-old patient with myelopathy underwent posterior cervical decompression and fusion, using rhBMP-2 as a bone graft substitute. The patient had complete resolution of symptoms after surgery until day 6, when she experienced increasing pain and weakness. T2 magnetic resonance images revealed a high intensity fluid collection compressing the cervical cord posteriorly. Emergent decompression was performed and the patient improved until postoperative day 12 when the same clinical scenario occurred. Symptoms again improved with surgical debridement. The clear, nonsanguineous fluid was sent for a quantitative cytokine panel each time. The case is reviewed with specific reference to the evolving literature regarding rhBMP-2 use in the spine, and the findings of seroma analysis. RESULTS: The fluid analysis of the seroma fluid at the time of both debridements showed impressive elevations in inflammatory cytokines, especially IL-6 and IL-8. CONCLUSION: Acute inflammatory reactions to rhBMP-2 can occur in the posterior cervical spine and can lead to significant morbidity. Host factors, BMP-2 dosage, and carrier factors all likely play a role in these complex reactions and must be considered every time an "off label" usage of rhBMP-2 is considered. More study is clearly indicated.

Immunohistochemical localization of cadherin and catenin adhesion molecules in the murine growth plate.

Mouse tibial growth plates were examined for the presence of adhesion molecules using immunohistochemistry and RT-PCR. All of the components of the classical cadherin/catenin complex (cadherin, alpha-, beta-, and gamma-catenin), as well as a heavy presence of p120, were identified in the murine growth plate. All of the major cadherins (1-5, 11, 13, and 15) were, for the first time, identified and localized in the murine growth plate. We have demonstrated that most of the cadherins and catenins reside in the zone of hypertrophy. Only alpha-catenin and E-, P-, R-, and VE-cadherin were found in all regions of the growth plate. The results for T-cadherin were inconclusive.

Alcohol and other factors affecting osteoporosis risk in women.

By about age 35, people reach their peak bone mass. Women lose bone mass slowly after that point until a few years after menopause, when bone mass is lost very rapidly. For middle-aged and older women, healthy bones depend on the development, during younger years, of a strong bone structure and an adequate peak bone mass. There is tenuous evidence that moderate alcohol consumption may protect bone. But human and animal studies clearly indicate that chronic heavy drinking, particularly during adolescence and the young adult years, can dramatically compromise bone quality and may increase osteoporosis risk. Further, research indicates that the effects of heavy alcohol use on bone cannot be reversed, even if alcohol consumption is terminated. Research suggests that in addition to alcohol, other lifestyle factors--such as tobacco use, nutrition, weight-bearing exercise, increased body weight, and hormone replacement therapy--affect bone development and osteoporosis risk in women. However, there has been little examination of how alcohol interacts with these factors to influence bone health.