Wnt10b regulates osteogenesis of adipose-derived stem cells through Wnt/ß-catenin signalling pathway in osteoporosis.

Our previous finding revealed that the Wnt10b RNA expression of osteoporotic adipose-derived stem cells (OP-ASCs) with impaired osteogenic capacity was significantly reduced than that of ASCs. There are no ideas that the relationship between the OP-ASCs' impaired osteogenic potential and Wnt10b expression. This study aimed to indicate the potential molecular mechanisms and functional role of Wnt10b in OP-ASCs, as well as to investigate a potential application to reverse the OP-ASCs' impaired osteogenic differentiation potential. The OP-ASCs and ASCs were harvested from the inguinal fat of osteoporosis (OP) mice with bilateral ovariectomy (OVX) and normal mice. qPCR and WB were used to detect the different levels of the expression of the Wnt10b RNA in both OP-ASCs and ASCs. Lentiviral-mediated regulation of Wnt10b expression was employed for OP-ASCs, and the detection of the expression levels of key molecules in the Wnt signalling pathway and key osteogenic factors was performed through qPCR and WB in vitro experiments. The capacity of OP-ASCs to osteogenesis was determined using alizarin red staining. Lastly, the repair effect of the BCP scaffolds incorporating modified OP-ASCs on the critical-sized calvarial defects (CSCDs) in OP mice was scanned and detected by micro-computed tomography, haematoxylin and eosin staining, Masson's trichrome staining and immunohistochemistry. First, we discovered that both the RNA and protein expression levels of Wnt10b were significantly lower in OP-ASCs than that in ASCs. In vitro experiments, upregulation of Wnt10b could activate the Wnt signalling pathway, and increase expression of ß-catenin, Lef1, Runx2 and osteopontin (Opn), thereby enhancing the osteogenic ability of OP-ASCs. In addition, the OP-ASCs with Wnt10b-overexpressing could promote the repair of CSCD in osteoporotic mice with increasing new bone volume, bone mineral density, and increased expression of Opn in new bone in vivo. Taken together, overexpression of Wnt10b could partially facilitate the differentiation of OP-ASCs towards osteogenesis and accelerated the healing of bone defects by activating the Wnt/ß-catenin signalling pathway in vitro and in vivo experiments. This study confirmed the important role of Wnt10b in regulating the osteogenic differentiation capability of OP-ASCs and indicated Wnt10b could be a potential therapeutic target for reversing the impaired osteogenic capabilities of OP-ASCs to therapy bone defects of OP patients.

A Biomimetic Se-nHA/PC Composite Microsphere with Synergistic Immunomodulatory and Osteogenic Ability to Activate Bone Regeneration in Periodontitis.

Accumulation of reactive oxygen species (ROS) in periodontitis exacerbates the destruction of alveolar bone. Therefore, scavenging ROS to reshape the periodontal microenvironment, alleviate the inflammatory response and promote endogenous stem cell osteogenic differentiation may be an effective strategy for treating bone resorption in periodontitis. In this study, sericin-hydroxyapatite nanoparticles (Se-nHA NPs) are synthesized using a biomimetic mineralization method. Se-nHA NPs and proanthocyanidins (PC) are then encapsulated in sericin/sodium alginate (Se/SA) using an electrostatic injection technique to prepare Se-nHA/PC microspheres. Microspheres are effective in scavenging ROS, inhibiting the polarization of macrophages toward the M1 type, and inducing the polarization of macrophages toward the M2 type. In normal or macrophage-conditioned media, the Se-nHA/PC microspheres effectively promoted the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Furthermore, the Se-nHA/PC microspheres demonstrated anti-inflammatory effects in a periodontitis rat model by scavenging ROS and suppressing pro-inflammatory cytokines. The Se-nHA/PC microspheres are also distinguished by their capacity to decrease alveolar bone loss, reduce osteoclast activity, and boost osteogenic factor expression. Therefore, the biomimetic Se-nHA/PC composite microspheres have efficient ROS-scavenging, anti-inflammatory, and osteogenic abilities and can be used as a multifunctional filling material for inflammatory periodontal tissue regeneration.

Spatiotemporal cellular dynamics and molecular regulation of tooth root ontogeny.

Tooth root development involves intricate spatiotemporal cellular dynamics and molecular regulation. The initiation of Hertwig's epithelial root sheath (HERS) induces odontoblast differentiation and the subsequent radicular dentin deposition. Precisely controlled signaling pathways modulate the behaviors of HERS and the fates of dental mesenchymal stem cells (DMSCs). Disruptions in these pathways lead to defects in root development, such as shortened roots and furcation abnormalities. Advances in dental stem cells, biomaterials, and bioprinting show immense promise for bioengineered tooth root regeneration. However, replicating the developmental intricacies of odontogenesis has not been resolved in clinical treatment and remains a major challenge in this field. Ongoing research focusing on the mechanisms of root development, advanced biomaterials, and manufacturing techniques will enable next-generation biological root regeneration that restores the physiological structure and function of the tooth root. This review summarizes recent discoveries in the underlying mechanisms governing root ontogeny and discusses some recent key findings in developing of new biologically based dental therapies.

Modified tragus edge incision and transmasseteric anteroparotid approach to condyle reconstruction.

OBJECTIVES: This study aimed to analyze the application value of a modified tragus edge incision and transmasseteric anteroparotid approach to condyle reconstruction. METHODS: Condyle reconstruction was performed in 16 patients (9 females and 7 males) with modified tragus edge incision and transmasseteric anteroparotid approach. After regular follow-up, the function of condyle reconstruction was evaluated by clinical indicators, such as parotid salivary fistula, facial nerve function, mouth opening, occlusal relationship, and facial scar. The morphology of rib graft rib cartilage was evaluated by imaging indicators, such as panoramic radiography, CT, and three-dimensional CT image reconstruction. RESULTS: At 6-36 months postoperative follow-up, all patients had good recovery of facial appearance, concealed incisional scar, no parotid salivary fistula, good mouth opening, and occlusion. One case had temporary facial paralysis and recovered after treatment. Radiographic evaluation further showed that costochondral graft survived in normal anatomic locations. CONCLUSIONS: The modified tragus edge incision and transmasseteric anteroparotid approach can effectively reduce parotid salivary fistula and facial nerve injury in condylar reconstruction. The surgical field was clearly exposed, and the incision scar was concealed without increasing the incidence of other complications. Thus, this approach is worthy of clinical promotion.

Methyltransferase-like 3 modulates osteogenic differentiation of adipose-derived stem cells in osteoporotic rats.

BACKGROUND: Osteoporosis (OP) is a metabolic bone disease involving reduced bone mass. Adipose-derived stem cells (ASCs) play an important role in bone regeneration. Emerging evidence suggests that methyltransferase-like 3 (METTL3) plays a significant role in bone development and metabolism. Therefore, this study investigates changes to METTL3 in the osteogenic differentiation of adipose stem cells in osteoporotic rats (OP-ASCs) and explores ways to enhance their osteogenic ability. METHODS: An animal model of osteoporosis was established by removing both ovaries in rats. Real-time PCR and western blotting were performed to detect the expression of METTL3 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Furthermore, alkaline phosphatase staining was used to confirm the osteogenic potential of stem cells. Mettl3 small interfering RNA and Mettl3 overexpression lentivirus were used to assess the role of METTL3 in osteogenic differentiation of ASCs and OP-ASCs. RESULTS: The osteogenic differentiation capacity and Mettl3 expression significantly decreased in OP-ASCs. Moreover, Mettl3 silencing down-regulated the osteogenic ability of ASCs, and overexpression of Mettl3 recovered the impaired osteogenic capacity in OP-ASCs in vitro. CONCLUSION: The Mettl3 expression levels and osteogenic potential of OP-ASCs decreased. However, overexpression of METTL3 rescued the osteogenic ability of OP-ASCs, providing a new target for treatment of osteoporotic bone defects.

Reconstruction of orbital wall fractures with superior orbital fissure syndrome using individualized preformed titanium mesh: a pilot study.

OBJECTIVE: The aim was to preliminarily evaluate the effect of individualized preformed titanium mesh in the treatment of orbital wall fractures with superior orbital fissure syndrome (SOFS). STUDY DESIGN: This study consisted of 10 patients of orbital wall fracture and SOFS who were treated at the Affiliated Stomatology Hospital of Southwest Medical University. On the basis of preoperative computed tomography data, individualized titanium mesh was produced by mirror engineering and rapid prototyping, and it was implanted into defects in the orbital walls to restore the normal anatomy. Early orbital wall reconstruction was performed to improve the SOFS. Postoperatively, the ocular and facial appearance and eye function were evaluated. RESULTS: The orbital structure, volume, and size of the SOF were restored in the 10 patients using the individualized titanium mesh. The symptoms of SOFS completely disappeared in all patients with no severe postoperative complications. Significant recovery of ocular and facial appearance and eye function was reported. CONCLUSIONS: This pilot study demonstrated that individualized preformed titanium mesh can accurately restore the orbital walls and the structure and size of the SOF, and it is useful in the treatment of SOFS without intraorbital bone fragment displacement.

Biomimetic Design and Fabrication of Sericin-Hydroxyapatite Based Membranes With Osteogenic Activity for Periodontal Tissue Regeneration.

The guided tissue regeneration (GTR) technique is a promising treatment for periodontal tissue defects. GTR membranes build a mechanical barrier to control the ingrowth of the gingival epithelium and provide appropriate space for the regeneration of periodontal tissues, particularly alveolar bone. However, the existing GTR membranes only serve as barriers and lack the biological activity to induce alveolar bone regeneration. In this study, sericin-hydroxyapatite (Ser-HAP) composite nanomaterials were fabricated using a biomimetic mineralization method with sericin as an organic template. The mineralized Ser-HAP showed excellent biocompatibility and promoted the osteogenic differentiation of human periodontal membrane stem cells (hPDLSCs). Ser-HAP was combined with PVA using the freeze/thaw method to form PVA/Ser-HAP membranes. Further studies confirmed that PVA/Ser-HAP membranes do not affect the viability of hPDLSCs. Moreover, alkaline phosphatase (ALP) staining, alizarin red staining (ARS), and RT-qPCR detection revealed that PVA/Ser-HAP membranes induce the osteogenic differentiation of hPDLSCs by activating the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN. The unique GTR membrane based on Ser-HAP induces the differentiation of hPDLSCs into osteoblasts without additional inducers, demonstrating the excellent potential for periodontal regeneration therapy.

Computed tomography angiography-aided individualized anterolateral thigh flap design in the reconstruction of oral and maxillofacial soft tissue defects.

PURPOSE: To evaluate a novel method and computed tomography angiography (CTA) for locating anterolateral thigh flap (ALTF) perforators to design individualized ALTFs to reconstruct maxillofacial soft tissue defects. STUDY DESIGN: This study comprised a group of 36 patients (CTA group) with malignant oral and maxillofacial tumors who underwent CTA and who received individualized ALTFs and a group of 28 patients (control group) with the same condition but without preoperative CTA examination and with nonindividualized ALTFs. ALTFs in the CTA group were designed and harvested using the locating device and CTA, whereas ALTFs in the control group were designed and harvested according to each surgeon's experience. RESULTS: Fifty perforators were located and 36 ALTFs harvested in the CTA group. In the control group, 34 perforators were located and 28 ALTFs harvested. Less time was required to locate the perforators in the CTA group. Moreover, the CTA group had a higher flap survival rate and better patient satisfaction regarding the postoperative aesthetics and phonetic and swallowing functions. CONCLUSIONS: The results suggest that CTA and the locating device can be used to accurately locate ALTF perforators and that this method aids in the design and harvesting of individualized ALTFs to achieve good functional and aesthetic outcomes.

Decreased autophagy impairs osteogenic differentiation of adipose-derived stem cells via Notch signaling in diabetic osteoporosis mice.

BACKGROUND: The osteogenic differentiation ability of adipose-derived stem cells (ASCs) is attenuated in type 2 diabetic osteoporosis (Dop) mice. Several studies suggest autophagy and Notch signaling pathway play vital roles in cell proliferation, differentiation, and osteogenesis. However, the mechanisms of autophagy and Notch signaling in the osteogenic differentiation of Dop ASCs were unclear. Thus, it is meaningful to reveal potential correlations between autophagy, Notch signaling, and osteogenesis, and explore involved molecular mechanisms in Dop ASCs. MATERIALS AND METHODS: The diabetic osteoporosis C57BL/6 mouse model, which was confirmed by micro-CT and HE & Masson staining, was established through high-sugar and high-fat diet and streptozotocin injection. ASCs were obtained from the inguinal subcutaneous fat of Dop mice. The multi-differentiation potential of ASCs was evaluated by staining with Alizarin Red (osteogenesis), Oil Red O (adipogenesis), and Alcian blue (chondrogenesis). Cell viability was assessed by Cell Counting Kit-8 assay. Torin1, an inhibitor of mTOR, was used to stimulate the autophagy signaling pathway. DAPT, a γ-secretase inhibitor, was used to suppress Notch signaling pathway activity. Gene and protein expression of autophagy, Notch signaling pathway, and osteogenic factors were detected by real-time quantitative PCR, western blot, and immunofluorescence microscopy. RESULTS: Our findings showed autophagy and osteogenic differentiation ability of Dop ASCs exhibited downward trends that were both rescued by Torin1. Notch signaling was suppressed in Dop ASCs, but upregulated when autophagy was activated. After activation of autophagy, DAPT treatment led to decreased Notch signaling pathway activation and attenuated osteogenic differentiation ability in Dop ASCs. CONCLUSIONS: Downregulated autophagy suppressed Notch signaling, leading to a reduced osteogenic differentiation capacity of Dop ASCs, and Torin1 can rescue this process by activating autophagy. Our findings contribute to understanding the mechanism underlying impairment of the osteogenic differentiation ability of Dop ASCs.

Accuracy evaluation of partially guided and fully guided templates applied to implant surgery of anterior teeth: A randomized controlled trial.

OBJECTIVES: To study the accuracy of partially guided and fully guided templates applied to implant surgery of anterior teeth. MATERIALS AND METHODS: Sixty patients who were scheduled to receive dental implant treatment in the anterior region were enrolled and randomly assigned to one of the following study groups (n = 20 each): routine implant-supported restoration treatment (control group, 30 implants), implant-supported restoration treatment using a partially guided template (test group 1, 36 implants), and implant-supported restoration treatment using a fully guided template (test group 2, 33 implants). The depth of implant was controlled for fully guided template. After implantation, planned implants and placed implants were superimposed using digital software, and the deviations (angular, coronal, apical, depth) were analyzed. Esthetic parameters were assessed at baseline, 6 months, and 1 year after the final restoration. Pink esthetic score (PES) and white esthetic score (WES) were respectively used to evaluate the soft tissue and restoration esthetic outcome. Each parameter of PES and WES is assessed with a 0-1-2 score with 2 being the best and 0 being the worst score. RESULTS: There were significant differences in all of the deviation parameters between the control group, test group 1, and test group 2 (p < 0.001). Mean angular, coronal, apical and depth deviations were all the highest in the control group (6.61 ± 1.09°, 1.05 ± 0.17 mm, 1.36 ± 0.13 mm, and 1.02 ± 0.13 mm, respectively), and lowest in test group 2 (2.05 ± 0.45°, 0.39 ± 0.12 mm, 0.28 ± 0.09 mm, and 0.24 ± 0.06 mm, respectively). At 1 year after the final restoration, the analysis revealed mean PES values of 7.09 ± 0.56 (control group), 8.39 ± 0.54 (test group 1), and 9.04 ± 0.35 (test group 2). The WES values were 7.24 ± 0.54 (control group), 8.47 ± 0.44 (test group 1), and 8.97 ± 0.38 (test group 2). At all examinations, the mean PES and WES values were both the highest in test group 2 and lowest in the control group. The PES and WES values recorded in the control group at baseline, 6 months, and 1 year after final restoration were significantly lower than those in test groups (p < 0.001). Moreover, the PES and WES values recorded in the test group 1 at baseline, 6 months, and 1 year after final restoration were significantly lower than those in test group 2 (p < 0.05). CONCLUSIONS: Digital surgical guides can improve the accuracy of the three-dimensional position of implants in the maxillary esthetic zone, the fully guided template has higher precision than that of the partially guided template, and plays an important role in obtaining the ideal esthetic outcome for maxillary anterior teeth.

Advanced glycation end products inhibit the osteogenic differentiation potential of adipose-derived stem cells by modulating Wnt/ß-catenin signalling pathway via DNA methylation.

OBJECTIVES: Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose-derived stem cells (ASCs) are widely used in the research of bone regeneration, the mechanisms of the osteogenic differentiation of ASCs from diabetic osteoporosis model remain unclear. This work aimed to investigate the influence and the molecular mechanisms of AGEs on the osteogenic potential of ASCs. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay was used to measure the change of AGEs in diabetic osteoporotic and control C57BL/6 mice. ASCs were obtained from the inguinal fat of C57BL/6 mice. AGEs, 5-aza2'-deoxycytidine (5-aza-dC) and DKK-1 were used to treat ASCs. Real-time cell analysis and cell counting kit-8 were used to monitor the proliferation of ASCs within and without AGEs. Real-time PCR, Western blot and Immunofluorescence were used to analyse the genes and proteins expression of osteogenic factors, DNA methylation factors and Wnt/ß-catenin signalling pathway among the different groups. RESULTS: The AGEs and DNA methylation were increased in the adipose and bone tissue of the diabetic osteoporosis group. Untreated ASCs had higher cell proliferation activity than AGEs-treatment group. The expression levels of osteogenic genes, Opn and Runx2, were lower, and mineralized nodules were less in AGEs-treatment group. Meanwhile, DNA methylation was increased, and the Wnt signalling pathway markers, including ß-Catenin, Lef1 and P-GSK-3ß, were inhibited. After treatment with 5-aza-dC, the osteogenic differentiation capacity of ASCs in the AGEs environment was restored and the Wnt signalling pathway was activated during this process. CONCLUSIONS: Advanced glycation end products inhibit the osteogenic differentiation ability of ASCs by activating DNA methylation and inhibiting Wnt/ß-catenin pathway in vitro. Therefore, DNA methylation may be promising targets for the bone regeneration of ASCs with diabetic osteoporosis.

[Precise investigation of digital guide plates applied to implant surgery of anterior teeth].

OBJECTIVE: To study the precision of digital guide plates applied to the implant surgery of anterior teeth. METHODS: Fifty patients scheduled to receive implant restoration treatment in anterior teeth were enrolled in this study and divided into two groups (n=25, each group): those who were given routine implant restoration treatment (control group, 45 implants) and those who received implant restoration treatment using a digital guide plate (test group, 51 implants). After implantation, planned and placed implants were superimposed using digital software, and deviations (corona, apex, depth, degree) were analyzed. Esthetic parameters were assessed at 1 week (baseline), 6 month, and 1 year post final restoration. Pink esthetic (PES) and white esthetic (WES) scores were respectively used to evaluate the soft tissue and restoration esthetic outcome. RESULTS: The deviation parameters in the test group were significantly lower than those in the control group (P<0.05). PES and WES values recorded for the control group at 1 week, 6 month, and 1 year post final restoration were significantly lower than those in the test group (P<0.05). CONCLUSIONS: The digital guide plate can improve the accuracy of the three-dimensional position of implants in the maxillary esthetic zone. As such, this device may play an important role in obtaining the ideal aesthetic effects of maxillary anterior teeth.

Functional Reconstruction of Mandibular Segment Defects With Individual Preformed Reconstruction Plate and Computed Tomographic Angiography-Aided Iliac Crest Flap.

PURPOSE: With the development of imaging technology and computer-assisted surgery in oral and maxillofacial surgery, digital technology is widely used in vascularized bone flap grafts for mandibular reconstruction. The aim of this study was to use digital technology throughout the treatment process to show that digital techniques can provide a reliable and accurate match between the mandible and the iliac crest flap to achieve functional reconstruction of mandibular segment defects. MATERIALS AND METHODS: Twenty patients underwent 3-dimensional (3D) computed tomography (CT), mirroring technology, 3D model prototyping, and CT angiography (CTA) for treatment planning. Individual preformed reconstruction plates were fabricated and iliac crest flaps were designed preoperatively. After complete resection of the mandibular lesion, the iliac crest flap was shaped to reconstruct the mandibular defects. RESULTS: During follow-up (range, 12 to 36 months), the facial shape, facial symmetry, and mouth opening of all patients recovered well. The 3D CT reconstruction also was evaluated for height, width, length, and bone healing of the iliac crest flap. Postoperative examination showed ideal bone union between the iliac crest flap and the mandible at 6 months. Nine patients received implant-supported fixed dentures to restore dentition. After follow-up, all patients were satisfied with their facial esthetics and function. The new mandible provided a suitable 3D position for implant-supported fixed partial dentures. CONCLUSION: Use of digital techniques throughout the course of treatment improves the predictability and convenience of functional mandibular reconstruction. Individual preformed reconstruction plates and CTA effectively guaranteed the accuracy of iliac flap preparation.

Blockade of receptors of advanced glycation end products ameliorates diabetic osteogenesis of adipose-derived stem cells through DNA methylation and Wnt signalling pathway.

OBJECTIVES: Diabetes mellitus-related osteoporosis is caused by the imbalance between bone absorption and bone formation. Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose-derived stem cells (ASCs) are promising adult stem cells in bone tissue regeneration, the ability of osteogenesis of ASCs in diabetic environment needs to explore. This study aimed to investigate the influence of AGEs on the osteogenic potential of ASCs and to explore the signalling pathways involved in its effect. MATERIALS AND METHODS: ASCs were isolated from inguinal fat and cultured in osteogenic media with or without AGEs and FPS-ZM1, an inhibitor of receptor for AGEs (RAGE). Alizarin red-S, Oil Red-O and Alcian blue staining were used to confirm osteogenic, adipogenic and chondrogenic potential of ASCs, respectively. Immunofluorescence, western blotting and real-time PCR were used to measure changes in markers of osteogenic differentiation, DNA methylation and Wnt signalling. RESULTS: The multipotentiality of ASCs was confirmed. Treated with AGEs, OPN and RUNX2 expressions of ASCs were reduced and there was a noticeable loss of mineralization, concomitant with an increase in the expression of RAGE, 5-MC, DNMT1 and DNMT3a. AGEs treatment also led to a loss of Wnt signalling pathway markers, including ß-Catenin and LEF1, with an increase in GSK-3ß. Treatment with the RAGE inhibitor, FPS-ZM1, rescued AGEs-induced loss of osteogenic potential, modulated DNA methylation and upregulated Wnt signalling in ASCs. CONCLUSIONS: Our results demonstrate that AGEs-RAGE signalling inhibits the osteogenic potential of ASCs under osteoinductive conditions by modulating DNA methylation and Wnt signalling. FPS-ZM1 can rescue the negative effects of AGEs and provide a possible treatment for bone tissue regeneration in patients with diabetic osteoporosis.

Cartilage Tissue Regeneration: The Roles of Cells, Stimulating Factors and Scaffolds.

Cartilage tissue engineering is an emerging technique for the regeneration of cartilage tissue damaged as a result of trauma or disease. As the propensity for healing and regenerative capabilities of articular cartilage are limited, its repair remains one of the most challenging issues of musculoskeletal medicine. Clinical treatments intended to promote the success and complete repair of partial- and fullthickness articular cartilage defects are still unpredictable. However, one of the most exciting theories is that treatment of damaged articular cartilage can be realized with cartilage tissue engineering. This notion has prompted tissue engineering research involving cells, stimulating factors and scaffolds, either alone or in combination. With these perspectives, this review aims to present a summary of cartilage tissue engineering including development, recent progress, and major steps taken toward the regeneration of functional cartilage tissue. In addition, we discussed the role of stimulating factors, including growth factors, gene therapies, biophysical stimuli, and bioreactors, as well as scaffolds, including natural, synthetic, and nanostructured scaffolds, in cartilage tissue regeneration. Special emphasis was placed on cell source, including chondrocytes, fibroblasts, and stem cells, as an important component of cartilage tissue engineering techniques. In conclusion, continued development of cartilage tissue engineering will support future applications for patients suffering from diseased cartilage tissue problems and osteoarthritis.

PPARγ and Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) arise from a variety of tissues, including bone marrow and adipose tissue and, accordingly, have the potential to differentiate into multiple cell types, including osteoblasts and adipocytes. Research on MSCs to date has demonstrated that a large number of transcription factors and ectocytic or intrastitial signaling pathways regulate adipogenic and osteogenic differentiation. A theoretical inverse relationship exists in adipogenic and osteogenic lineage commitment and differentiation, such that signaling pathways induce adipogenesis at the expense of osteogenesis and vice versa. For example, peroxisome proliferator-activated receptor γ(PPARγ), which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors, is known to function as a master transcriptional regulator of adipocyte differentiation, and inhibit osteoblast differentiation. Moreover, recent studies have demonstrated that inducers of osteogenic differentiation, such as bone morphogenetic protein (BMP) and Wnt, inhibit the function of PPARγ transactivation during MSC differentiation towards adipocytes through a variety of mechanisms. To illustrate this, the canonical Wnt/ß-catenin pathway represses expression of PPARγ mRNA, whereas the noncanonical Wnt pathway activates histone methyltransferases that inhibit PPARγ transactivation via histone H3 lysine 9 (H3K9) methylation of its target genes. The role of microRNAs (miRNAs) in adipogenesis and osteoblastogenesis is garnering increased attention, and studies in this area have shed light on the integration of miRNAs with Wnt signaling and transcription factors such as Runx2 and PPARγ. This review summarizes our current understanding of the mechanistic basis of these signaling pathways, and indicates future clinical applications for stem cell-based cell transplantation and regenerative therapy.

Distinct Etiological Roles for Myocytes and Motor Neurons in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy.

Polyglutamine (polyQ) expansion of the androgen receptor (AR) causes Kennedy's disease/spinobulbar muscular atrophy (KD/SBMA) through poorly defined cellular mechanisms. Although KD/SBMA has been thought of as a motor neuron disease, recent evidence indicates a key role for skeletal muscle. To resolve which early aspects of the disease can be caused by neurogenic or myogenic mechanisms, we made use of the tet-On and Cre-loxP genetic systems to selectively and acutely express polyQ AR in either motor neurons (NeuroAR) or myocytes (MyoAR) of transgenic mice. After 4 weeks of transgene induction in adulthood, deficits in gross motor function were seen in NeuroAR mice, but not MyoAR mice. Conversely, reduced size of fast glycolytic fibers and alterations in expression of candidate genes were observed only in MyoAR mice. Both NeuroAR and MyoAR mice exhibited reduced oxidative capacity in skeletal muscles, as well as a shift in fast fibers from oxidative to glycolytic. Markers of oxidative stress were increased in the muscle of NeuroAR mice and were reduced in motor neurons of both NeuroAR and MyoAR mice. Despite secondary pathology in skeletal muscle and behavioral deficits, no pathological signs were observed in motor neurons of NeuroAR mice, possibly due to relatively low levels of polyQ AR expression. These results indicate that polyQ AR in motor neurons can produce secondary pathology in muscle. Results also support both neurogenic and myogenic contributions of polyQ AR to several acute aspects of pathology and provide further evidence for disordered cellular respiration in KD/SBMA skeletal muscle.

Microarray analysis of gene expression by skeletal muscle of three mouse models of Kennedy disease/spinal bulbar muscular atrophy.

BACKGROUND: Emerging evidence implicates altered gene expression within skeletal muscle in the pathogenesis of Kennedy disease/spinal bulbar muscular atrophy (KD/SBMA). We therefore broadly characterized gene expression in skeletal muscle of three independently generated mouse models of this disease. The mouse models included a polyglutamine expanded (polyQ) AR knock-in model (AR113Q), a polyQ AR transgenic model (AR97Q), and a transgenic mouse that overexpresses wild type AR solely in skeletal muscle (HSA-AR). HSA-AR mice were included because they substantially reproduce the KD/SBMA phenotype despite the absence of polyQ AR. METHODOLOGY/PRINCIPAL FINDINGS: We performed microarray analysis of lower hindlimb muscles taken from these three models relative to wild type controls using high density oligonucleotide arrays. All microarray comparisons were made with at least 3 animals in each condition, and only those genes having at least 2-fold difference and whose coefficient of variance was less than 100% were considered to be differentially expressed. When considered globally, there was a similar overlap in gene changes between the 3 models: 19% between HSA-AR and AR97Q, 21% between AR97Q and AR113Q, and 17% between HSA-AR and AR113Q, with 8% shared by all models. Several patterns of gene expression relevant to the disease process were observed. Notably, patterns of gene expression typical of loss of AR function were observed in all three models, as were alterations in genes involved in cell adhesion, energy balance, muscle atrophy and myogenesis. We additionally measured changes similar to those observed in skeletal muscle of a mouse model of Huntington's Disease, and to those common to muscle atrophy from diverse causes. CONCLUSIONS/SIGNIFICANCE: By comparing patterns of gene expression in three independent models of KD/SBMA, we have been able to identify candidate genes that might mediate the core myogenic features of KD/SBMA.

Myocyte androgen receptors increase metabolic rate and improve body composition by reducing fat mass.

Testosterone and other androgens are thought to increase lean body mass and reduce fat body mass in men by activating the androgen receptor. However, the clinical potential of androgens for improving body composition is hampered by our limited understanding of the tissues and cells that promote such changes. Here we show that selective overexpression of androgen receptor in muscle cells (myocytes) of transgenic male rats both increases lean mass percentage and reduces fat mass. Similar changes in body composition are observed in human skeletal actin promoter driving expression of androgen receptor (HSA-AR) transgenic mice and result from acute testosterone treatment of transgenic female HSA-AR rats. These shifts in body composition in HSA-AR transgenic male rats are associated with hypertrophy of type IIb myofibers and decreased size of adipocytes. Metabolic analyses of transgenic males show higher activity of mitochondrial enzymes in skeletal muscle and increased O(2) consumption by the rats. These results indicate that androgen signaling in myocytes not only increases muscle mass but also reduces fat body mass, likely via increases in oxidative metabolism.

A tetracycline-inducible and skeletal muscle-specific Cre recombinase transgenic mouse.

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline-controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP-LacZ reporter mice, which express beta-galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice.