Saqib Rehman

Molecular Requirements for Induction of CTGF Expression by TGF-β1 in Primary Osteoblasts

Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-beta1 where it acts as a downstream mediator of TGF-beta1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-beta1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-beta1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-beta1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-beta1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-beta1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-beta1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-beta1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-beta in some cell types, we determined its role in TGF-beta1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-beta1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-beta1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To investigate the involvement of the TGF-beta1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction, we cloned the rat CTGF proximal promoter (-787 to +1) containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using a combination of CTGF promoter deletion constructs and site-directed mutants, we demonstrated the unique requirement of both the TRE and SBE for CTGF induction by TGF-beta1 in osteoblasts. Electro-mobility shift assays using specific probes containing the TRE, SBE or both showed TGF-beta1 inducible complexes that can be ablated by mutation of the respective motif, confirming their requirement for TGF-beta1 induced CTGF promoter activity. In conclusion, these studies demonstrate that CTGF induction by TGF-beta1 in osteoblasts involves Smads 3 and 4, the Erk and Src signaling pathways, and requires both the TRE and SBE motifs in the CTGF proximal promoter.

Temporal and spatial expression of osteoactivin during fracture repair

We previously identified osteoactivin (OA) as a novel secreted osteogenic factor with high expression in developing long bones and calvaria, and that stimulates osteoblast differentiation and matrix mineralization in vitro. In this study, we report on OA mRNA and protein expression in intact long bone and growth plate, and in fracture calluses collected at several time points up to 21 days post‐fracture (PF). OA mRNA and protein were highly expressed in osteoblasts localized in the metaphysis of intact tibia, and in hypertrophic chondrocytes localized in growth plate, findings assessed by in situ hybridization and immunohistochemistry, respectively. Using a rat fracture model, Northern blot analysis showed that expression of OA mRNA was significantly higher in day‐3 and day‐10 PF calluses than in intact rat femurs. Using in situ hybridization, we examined OA mRNA expression during fracture healing and found that OA was temporally regulated, with positive signals seen as early as day‐3 PF, reaching a maximal intensity at day‐10 PF, and finally declining at day‐21 PF. At day‐5 PF, which correlates with chondrogenesis, OA mRNA levels were significantly higher in the soft callus than in intact femurs. Similarly, we detected high OA protein immunoexpression throughout the reparative phase of the hard callus compared to intact femurs. Interestingly, the secreted OA protein was also detected within the newly made cartilage matrix and osteoid tissue. Taken together, these results suggest the possibility that OA plays an important role in bone formation and serves as a positive regulator of fracture healing. J. Cell. Biochem. 111: 295–309, 2010.

Src is a major signaling component for CTGF induction by TGF-β1 in osteoblasts†

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor β1 (TGF‐β1) where it acts as a downstream mediator of TGF‐β1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF‐β1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF‐β1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF‐β1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF‐β1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co‐activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF‐β1‐induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF‐β1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. J. Cell. Physiol. 224: 691–701, 2010.

The importance of surgical sequence in the treatment of lower extremity injuries with concomitant vascular injury: A meta-analysis

OBJECTIVE The optimal sequence of surgical repair for lower extremity injury with associated vascular injuries is unclear. Lower extremity injury in our study is defined as femoral fracture, tibial fracture, and/or knee dislocation. Advocates of performing the vascular repair prior to lower extremity fixation argue that reversal of ischaemia in the limb is the most important factor in limb survival and should take precedence. Advocates of lower extremity fixation prior to revascularisation worry that the manipulation during fixation could disrupt the vascular repair and that total ischaemia time is more relative than absolute. METHODS A literature search was performed to identify studies with the following criteria: adult population, femoral fracture, tibial fracture, and/or knee dislocation with associated vascular injury, an intervention of fracture fixation or knee stabilisation prior to revascularisation and/or revascularisation prior to fracture fixation, and amputation as an outcome measurement. RESULTS 934 articles were identified and narrowed to 14 articles through exclusion criteria. Meta-analysis of the data shows no statistical difference in regards to the incidence of amputation between lower extremity fixation prior to revascularisation and revascularisation prior to fracture fixation. CONCLUSION Lower extremity injuries with associated vascular injury are uncommon. There has been a widespread but unsupported belief that manipulation and traction during lower extremity fixation will disrupt the vascular repair. Ischaemic time should be considered a relative, but not absolute predictor of amputation. Soft tissue injury and neurologic deficits have been found highly correlated with disability and amputation. Surgical sequence has not been shown to affect the rate of amputations in lower extremity fractures.

Intramedullary Fixation of Forearm Fractures

Plate fixation remains the primary surgical treatment option for most adult forearm fractures. However, intramedullary nailing can be successful and might be preferable in cases of massive soft-tissue injury and burns, certain segmental fractures, pathologic fractures, and skeletally immature adolescent patients. Furthermore, the risk for refracture after plate removal is decreased with fixation by intramedullary nailing. The history, indications, surgical technique, and results of intramedullary fixation of forearm fractures are described in this article.

Percutaneous Sacroiliac Screw Fixation of the Posterior Pelvic Ring

Percutaneous sacroiliac (SI) screw fixation is indicated for unstable posterior pelvic ring injuries, sacral fractures, and SI joint dislocations. This article provides a review of indications and contraindications, preoperative planning, imaging techniques and relevant anatomy, surgical technique, complications and their management, and outcomes after SI screw insertion.

Traumatic Knee Dislocations: Evaluation, Management, and Surgical Treatment

Knee dislocations are catastrophic injuries that demand emergent evaluation and often require a multidisciplinary approach. Long-term outcome studies are relatively scarce secondary to the variability in any given study population and the wide variety of injury patterns between knee dislocations. Multiple controversies exist with regard to outcomes using various treatment methods (early vs late intervention, graft selection, repair vs reconstruction of medial and lateral structures, rehabilitation regimens). Careful clinical evaluation is essential when knee dislocation is suspected.

Disparities in internet use among orthopedic outpatients.

Internet access has lagged behind for patients with lower incomes and from certain ethnic groups. This study investigated the possible improvement of access to health-related information on the Internet for all patients in an urban outpatient setting, regardless of socioeconomic background. A 28-question survey was completed by 100 orthopedic outpatients evaluating associations between their age, ethnicity, income, or education level and their access to the Internet. The survey also examined how patients used the Internet to obtain information about their medical condition, their privacy concerns when conducting online research, and their use of mobile phones as a primary means of Internet access. The Internet was used by 57% of orthopedic outpatients in this urban setting. Internet access decreased with advancing age but increased with increasing income and education, findings consistent with similar studies. Despite the inability to identify an association between ethnicity and Internet access in this patient population, fewer Latinos (33%) than whites (67%) or African Americans (77%) sought information about their medical condition. Among patients who used a mobile phone as the primary method for online access, 74% were African American or Latino and 26% were white. This difference in mobile phone use for online access suggests that mobile phones have provided ethnic minorities with greater Internet access and thus may have narrowed the digital divide among the races.

Management of Traumatic Bone Loss in the Lower Extremity

Segmental bone loss represents a difficult clinical entity for the treating orthopedic surgeon. This article discusses the various treatment modalities available for limb reconstruction, with a focus on the indications, potential complications, and the outcomes of available treatment options.

Biomechanical comparison of locked plating and spiral blade retrograde nailing of supracondylar femur fractures

OBJECTIVE Biomechanical comparison between locked plating and retrograde nailing of supracondylar femur fractures with simulated postoperative weight-bearing. METHODS The Locking Condylar Plate (LCP) and Retrograde/Antegrade EX Femoral Nail (RAFN) were tested using 10 paired elderly cadaveric femurs, divided into Normal and Low Bone Mineral Density (BMD) groups, with a simulated AO/OTA type 33-A3 supracondylar femur fracture. Each specimen was subjected to 200,000 loading cycles in an attempt to simulate six weeks of postoperative recovery with full weight-bearing for an average individual. The constructs subsidence due to cyclic loading, and axial stiffness before and after the cyclic loading were measured and their correlation with BMD was studied. The two implants were compared in a paired study within each BMD group. RESULTS LCP constructs showed higher axial stiffness compared to RAFN for both Normal and Low BMD groups (80% and 57%, respectively). After cyclic loading, axial stiffness of both constructs decreased by 20% and RAFN constructs resulted in twice as much subsidence (1.9 ± 0.6mm). Two RAFN constructs with Low BMD failed after a few cycles whereas the matched pairs fixed with LCP failed after 70,000 cycles. CONCLUSIONS The RAFN constructs experienced greater subsidence and reduced axial stiffness compared to the LCP constructs. In Low BMD specimens, the RAFN constructs had a higher risk of failure.