Lewis L. Shi

Bone Morphogenetic Protein (BMP) signaling in development and human diseases.

Bone Morphogenetic Proteins (BMPs) are a group of signaling molecules that belongs to the Transforming Growth Factor-β (TGF-β) superfamily of proteins. Initially discovered for their ability to induce bone formation, BMPs are now known to play crucial roles in all organ systems. BMPs are important in embryogenesis and development, and also in maintenance of adult tissue homeostasis. Mouse knockout models of various components of the BMP signaling pathway result in embryonic lethality or marked defects, highlighting the essential functions of BMPs. In this review, we first outline the basic aspects of BMP signaling and then focus on genetically manipulated mouse knockout models that have helped elucidate the role of BMPs in development. A significant portion of this review is devoted to the prominent human pathologies associated with dysregulated BMP signaling.

The versatile functions of Sox9 in development, stem cells, and human diseases

The transcription factor Sox9 was first discovered in patients with campomelic dysplasia, a haploinsufficiency disorder with skeletal deformities caused by dysregulation of Sox9 expression during chondrogenesis. Since then, its role as a cell fate determiner during embryonic development has been well characterized; Sox9 expression differentiates cells derived from all three germ layers into a large variety of specialized tissues and organs. However, recent data has shown that ectoderm- and endoderm-derived tissues continue to express Sox9 in mature organs and stem cell pools, suggesting its role in cell maintenance and specification during adult life. The versatility of Sox9 may be explained by a combination of post-transcriptional modifications, binding partners, and the tissue type in which it is expressed. Considering its importance during both development and adult life, it follows that dysregulation of Sox9 has been implicated in various congenital and acquired diseases, including fibrosis and cancer. This review provides a summary of the various roles of Sox9 in cell fate specification, stem cell biology, and related human diseases. Ultimately, understanding the mechanisms that regulate Sox9 will be crucial for developing effective therapies to treat disease caused by stem cell dysregulation or even reverse organ damage.

Semiconstrained Primary and Revision Total Elbow Arthroplasty with Use of the Coonrad-Morrey Prosthesis

BACKGROUND Semiconstrained total elbow prostheses are used routinely by many surgeons to treat a variety of severe elbow disorders. Our objective was to review the results of primary and revision total elbow arthroplasty with use of the Coonrad-Morrey prosthesis. The selected use of this semiconstrained implant in patients with instability and poor bone stock was hypothesized to provide inferior results compared with those in the published reports. METHODS The results of sixty-seven semiconstrained total elbow arthroplasties that were performed in fifty-six patients between 1990 and 2003 were evaluated. Thirty-seven elbows had a primary arthroplasty and were followed for a mean of eighty-six months, and thirty elbows had a revision arthroplasty and were followed for a mean of sixty-eight months. Mayo elbow performance scores and radiographic analyses were used to assess the clinical results. RESULTS In the primary arthroplasty group, the average flexion improved from 116 degrees to 135 degrees; average extension, from -40 degrees to -33 degrees; average pronation, from 60 degrees to 81 degrees; and average supination, from 60 degrees to 69 degrees. The improvements in flexion and pronation were significant (p<0.001 for both). Preoperatively, twenty-five (74%) of thirty-four elbows with data available had moderate or severe pain, whereas only four (11%) had pain postoperatively. The average postoperative Mayo score (and standard deviation) was 84+/-16. Eleven of the thirty-seven primary replacements failed, and the five-year survival rate was 72%. In the revision arthroplasty group, average flexion improved from 124 degrees to 131 degrees; average extension, from -32 degrees to -22 degrees; average pronation, from 66 degrees to 75 degrees; and average supination, from 64 degrees to 76 degrees; the improvement in supination was significant (p<0.05). Preoperatively, eighteen (64%) of the twenty-eight elbows with data available had moderate or severe pain, while only five (17%) had pain postoperatively. The average postoperative Mayo score was 85+/-16. Eleven of the thirty revision replacements failed, and the five-year survival rate was 64%. CONCLUSIONS A Coonrad-Morrey semiconstrained total elbow arthroplasty provides excellent pain relief and good functional return in patients with severe destructive arthropathy. The higher prevalence of failure in this cohort compared with series reported elsewhere is likely due to adverse patient selection as this implant was reserved for more complex arthroplasties with severe bone loss and ligamentous laxity.

Wnt/β-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance

Wnt signaling transduces evolutionarily conserved pathways which play important roles in initiating and regulating a diverse range of cellular activities, including cell proliferation, calcium homeostasis, and cell polarity. The role of Wnt signaling in controlling cell proliferation and stem cell self-renewal is primarily carried out through the canonical pathway, which is the best-characterized the multiple Wnt signaling branches. The past 10 years has seen a rapid expansion in our understanding of the complexity of this pathway, as many new components of Wnt signaling have been identified and linked to signaling regulation, stem cell functions, and adult tissue homeostasis. Additionally, a substantial body of evidence links Wnt signaling to tumorigenesis of cancer types and implicates it in the development of cancer drug resistance. Thus, a better understanding of the mechanisms by which dysregulation of Wnt signaling precedes the development and progression of human cancer may hasten the development of pathway inhibitors to augment current therapy. This review summarizes and synthesizes our current knowledge of the canonical Wnt pathway in development and disease. We begin with an overview of the components of the canonical Wnt signaling pathway and delve into the role this pathway has been shown to play in stemness, tumorigenesis, and cancer drug resistance. Ultimately, we hope to present an organized collection of evidence implicating Wnt signaling in tumorigenesis and chemoresistance to facilitate the pursuit of Wnt pathway modulators that may improve outcomes of cancers in which Wnt signaling contributes to aggressive disease and/or treatment resistance.

Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9‐induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9‐induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre‐osteoblast C2C12 cells. BMP9‐induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7‐induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9‐induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2‐induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9‐induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9s potent osteogenic capability in MSCs.

Crosstalk between Wnt/β-catenin and estrogen receptor signaling synergistically promotes osteogenic differentiation of mesenchymal progenitor cells.

Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.

Surgical treatment of isolated type II superior labrum anterior-posterior (SLAP) lesions: repair versus biceps tenodesis

BACKGROUND It is still unclear which patients with isolated type II superior labrum anterior-posterior (SLAP) lesions benefit from either superior labral repair or biceps tenodesis. This study evaluates the indications and outcomes of patients with isolated type II SLAP lesions who have undergone either procedure. METHODS A retrospective analysis was performed of patients who had surgery for an isolated type II SLAP lesion between 2008 and 2011. There were 25 patients: 15 underwent biceps tenodesis, with a mean follow-up of 31 months (range, 26-43 months), and 10 underwent SLAP repair, with a mean follow-up of 35 months (range, 25-52 months). The mean age was 47 years (range, 30-59 years) in the tenodesis group and 31 years (range, 21-43 years) in the repair group. RESULTS At latest follow-up, both groups showed significant improvements in subjective shoulder value and pain score. No difference was observed in American Shoulder and Elbow Surgeons score (93.0 vs 93.5, P = .45), patient satisfaction (93% vs 90%, P = .45), or return to preinjury sporting level (73% vs 60%, P = .66). Analysis of the indications for treatment showed that in the large majority, tenodesis was performed in older patients (>35 years) and patients who showed degenerative or frayed labrums whereas SLAP repairs were performed in younger and more active patients with healthy-appearing labral tissue. There was only 1 failure in the tenodesis group, and in the SLAP repair group, there were 2 cases of postoperative stiffness; all were treated nonoperatively. CONCLUSION In this study, we show that both biceps tenodesis and SLAP repair can provide good to excellent results if performed in appropriately selected patients with isolated type II SLAP lesions.

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.

The piggyBac Transposon-Mediated Expression of SV40 T Antigen Efficiently Immortalizes Mouse Embryonic Fibroblasts (MEFs)

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs’ basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.

Bioresorbable polylactide interbody implants in an ovine anterior cervical discectomy and fusion model: three-year results.

Study Design. In vivo study of anterior discectomy and fusion using a bioresorbable 70:30 poly(l-lactide-co-d,l-lactide) interbody implant in an ovine model. Objective. To evaluate the efficacy of the polylactide implant to function as an interbody fusion device, and to assess the tissue reaction to the material during the resorption process. Summary of Background Data. The use of polylactide as a cervical interbody implant has several potential advantages when compared with traditional materials. Having an elastic modulus very similar to bone minimizes the potential for stress shielding, and as the material resorbs additional loading is transferred to the developing fusion mass. Although preclinical and clinical studies have demonstrated the suitability of polylactide implants for lumbar interbody fusion, detailed information on cervical anterior cervical discectomy and fusion (ACDF) with polylactide devices is desirable. Methods. Single level ACDF was performed in 8 skeletally mature ewes. Bioresorbable 70:30 poly (l-lactide-co-d,l-lactide) interbody implants packed with autograft were used with single-level metallic plates. Radiographs were made every 3 months up to 1 year, and yearly thereafter. The animals were killed at 6 months (3 animals), 12 months (3 animals), and 36 months (2 animals). In addition to the serial plain radiographs, the specimens were evaluated by nondestructive biomechanical testing and undecalcified histologic analysis. Results. The bioresorbable polylactide implants were effective in achieving interbody fusion. The 6-month animals appeared fused radiographically and biomechanically, whereas histologic sections demonstrated partial fusion (in 3 of 3 animals). Radiographic fusion was confirmed histologically and biomechanically at 12 months (3 of 3 animals) and 36 months (2 of 2 animals). A mild chronic inflammatory response to the resorbing polylactide implant was observed at both 6 months and 12 months. At 36 months, the operative levels were solidly fused and the implants were completely resorbed. No adverse tissue response was observed in any animal at any time period. Conclusion. Interbody fusion was achieved using bioresorbable polylactide implants, with no evidence of implant collapse, extrusion, or adverse tissue response to the material. The use of polylactide as a cervical interbody device appears both safe and effective based on these ACDF animal model results.