Proliferation Of Bone Marrow Stem Cells Research Articles

3-Dimensional Bioprinting of a Tendon Stem Cell–Derived Exosomes Loaded Scaffold to Bridge the Unrepairable Massive Rotator Cuff Tear

Background: Unrepairable massive rotator cuff tears (UMRCTs) are challenging to surgeons owing to the severely retracted rotator cuff musculotendinous tissues and extreme defects in the rotator cuff tendinous tissues. Purpose: To fabricate a tendon stem cell–derived exosomes loaded scaffold (TSC-Exos-S) and investigate its effects on cellular bioactivity in vitro and repair in a rabbit UMRCT model in vivo. Study Design: Controlled laboratory study. Methods: TSC-Exos-S was fabricated by loading TSC-Exos and type 1 collagen (COL-I) into a 3-dimensional bioprinted and polycaprolactone (PCL)–based scaffold. The proliferation, migration, and tenogenic differentiation activities of rabbit bone marrow stem cells (BMSCs) were evaluated in vitro by culturing them in saline, PCL-based scaffold (S), COL-I loaded scaffold (COL-I-S), and TSC-Exos-S. In vivo studies were conducted on a rabbit UMRCT model, where bridging was repaired with S, COL-I-S, TSC-Exos-S, and autologous fascia lata (FL). Histological and biomechanical analyses were performed at 8 and 16 weeks postoperatively. Results: TSC-Exos-S exhibited reliable mechanical strength and subcutaneous degradation, which did not occur before tissue regeneration. TSC-Exos-S significantly promoted the proliferation, migration, and tenogenic differentiation of rabbit BMSCs in vitro. In vivo studies showed that UMRCT repaired with TSC-Exos-S exhibited significant signs of tendinous tissue regeneration at the bridging site with regard to specific collagen staining. Moreover, no significant differences were observed in the histological and biomechanical properties compared with those repaired with autologous FL. Conclusion: TSC-Exos-S achieved tendinous tissue regeneration in UMRCT by providing mechanical support and promoting the trend toward tenogenic differentiation. Clinical Relevance: The present study proposes a potential strategy for repairing UMRCT with severely retracted musculotendinous tissues and large tendinous tissue defects.

Extraction, Characterization and Osteogenic Activity of a Type I Collagen from Starfish (Asterias amurensis).

Outbreaks of starfish (Asterias amurensis) pose a major threat to aquaculture and marine ecosystems in Qingdao, China, and no effective methods have been found to control them. A comprehensive study of collagen in starfish could be an alternative to high efficient utilization. Based on this, collagen was firstly extracted from Qingdao A. amurensis. Then, its protein pattern, amino acid composition, secondary structure, microstructure and thermal stability were investigated. The results showed that the A. amurensis collagen (AAC) is a type I collagen composed of α1, α2, and β chains. Glycine, hydroxyproline, and alanine were the major amino acids. The melting temperature was 57.7 °C. From FTIR, UV spectra and CD chromatography, the AAC had an intact triple helix and secondary structure, and microstructural analysis showed that the AAC had a loose, fibrous porous structure. Next, the osteogenic differentiation effect of AAC on Mouse bone marrow stem cells (BMSCs) was investigated, and the results showed that AAC induced osteogenic differentiation of cells by promoting the proliferation of BMSCs, enhancing alkaline phosphatase (ALP) activity, promoting cell mineralization nodules and upregulating the expression of mRNA of relevant osteogenic genes. These results suggest that AAC might have the potential application to bone health-related functional foods.

Chordin-Like 1 Regulates Bone Marrow Stem Cell Proliferation and Excretion of Exosome and Myocardial Damage Under High Sugar

This study assesses the role of CHRDL1 (Chordin-like 1) i in the bone marrow stromal cells (BMSC) proliferation and excretion of exosome and myocardial damage under high sugar. BMSC from rats with high CHRDL1 expression was established. The exosome in the supernatant of BMSC with high CHRDL1 level was collected. H9C2 cells were assigned into control set, high sugar set and exo-CHRDL1-BMSC set followed by analysis of CHRDL1 level, BMSC proliferation and apoptosis, expression of Beclin-1, Atg5, Bcl-2 and Bax, and ROS and SOD activity. Cell proliferation was prompted and apoptotic activity was reduced in exo-CHRDL1-BMSC set with reduced ROS activity and increased SOD activity as well as upregulated Bcl-2 and downregulated Bax. In addition, exo-CHRDL1-BMSC set presented increased CHRDL1 secretion and upregulated Beclin-1 and Atg5 expression. In conclusion, proliferation of BMSC under high sugar is prompted and apoptosis is reduced by CHRDL1 through regulating the autophagy.

Graphene oxide coated three-dimensional printed biphasic calcium phosphate scaffold for angiogenic and osteogenic synergy in repairing critical-size bone defect

The custom-tailored medicine requires a developmental strategy that integrates excellent osteogenesis with mechanical stability to enhance the reconstruction of the critical-size bone defect (CSBD) and the healing process in weight-bearing bone. We prepared three-dimensional (3D) printed biphasic calcium phosphate (BCP) scaffolds composited with nano-graphene oxide (GO). The biological effects of the GO/BCP composite scaffolds could induce the differentiation of rat bone marrow stem cells (BMSCs) and the migration of human umbilical vein endothelial cells (HUVECs) for bone repair. The proper ratio of GO in the composite scaffold regulated the composites’ surface roughness and hydrophilicity to a suitable range for the adhesion and proliferation of BMSCs and HUVECs. Besides, the GO/BCP composite scaffold increased osteogenesis and angiogenesis by activating BMP-2, RUNX-2, Smad1/4, and VEGF. The customized intramedullary nail combined with GO/BCP scaffold was applied to repair CSBD (2.0 cm in length) in a beagle femur model. This fixation strategy was confirmed by finite element analysis. In vivo, the results indicated that the custom-made internal fixation provided sufficient stability in the early stage, ensuring bone healing in a considerable mechanical environment. At 9 months postoperatively, longitudinal bony union and blood vessels in osteon were observed in the CSBD area with partial degradation in the 0.3% GO/BCP group. In the three-point bending test, the ultimate load of 0.3% GO/BCP group reached over 50% of the normal femur at 9 months after repair. These results showed a promising application of osteogenic GO/BCP scaffold and custom-made intramedullary nails in repairing CSBD of the beagle femur. This effective strategy could provide an option to treat the clinical CSBD in weight-bearing bones.

Decreased Ankyrin Expression Is Associated with Repressed eNOS Signaling, Cell Proliferation, and Osteogenic Differentiation in Osteonecrosis of the Femoral Head.

Reduced nitric oxide synthase (NOS) activity and decreased reparative potentials in stem cells may be involved in the pathogenesis of osteonecrosis of the femoral head (ONFH), but the underlying mechanism is not clear. Ankyrin, a cytoskeletal protein, can promote NOS expression and many cellular functions when it interacts with the CD44 receptors on the stem cells. This study investigated whether ankyrin is involved in the pathogenesis of ONFH. Bone marrow stem cells (BMSCs) from ONFH patients were compared with cells from patients with proximal femoral fracture and BMSC cell lines (PT-2501, Lonza, NC, USA). Differences in the expression levels and downstream signal pathway of ankyrin-Akt-eNOS in BMSCs were studied between ONFH and control. The involvement of ankyrin in the signal cascade, cell proliferation, and differentiation were further investigated by silencing ankyrin using small interfering (si)RNA. We found the basal mRNA levels of ankyrin and CD44 in BMSCs from the ONFH group were significantly lower as compared with those from the control group. The signal transduction of CD44-ankyrin-Akt-eNOS was significantly repressed in the ONFH group as compared with the control group after hyaluronic acid treatment. Knockdown of ankyrin by siRNA could attenuate the eNOS signaling as well as the BMSCs proliferation and osteogenic differentiation. The proliferation ability and osteogenic differentiation potential of the BMSCs from the ONFH group were significantly reduced as compared with the control group, but they can be enhanced to the baseline levels of the control group by hyaluronic acid treatment. The aberrant eNOS signaling, reduced cell proliferation, and osteogenic differentiation potential in BMSCs from ONFH patients are associated with the decreased ankyrin expression. Altered signal transduction, proliferation, and osteogenic differentiation ability in BMSCs may be involved in the pathogenesis of ONFH. These need further studies especially in BMSC-based cell therapy.

LncRNA FGD5-AS1 Regulates Bone Marrow Stem Cell Proliferation and Apoptosis by Affecting miR-296-5p/STAT3 Axis in Steroid-Induced Osteonecrosis of the Femoral Head.

Background Osteonecrosis of the femoral head (ONFH) is a common hip joint disease, which is more harmful and seriously affects the lives of patients. This study aims to clarify the regulatory mechanism of lncRNA FGD5-AS1 in ONFH. Methods The expression of the protein and mRNA was detected by RT-qPCR and Western blot assay. The regulatory mechanism of lncRNA FGD5-AS1 was detected by the dual-luciferase reporter assay, CCK-8 assay, and flow cytometry assay. Results Dex can inhibit cell proliferation and differentiation and induce apoptosis in hBMSCs in a dose-dependent manner. Overexpression of lncRNA FGD5-AS1 promoted cell proliferation and restrained apoptosis in Dex-treated hBMSCs. In addition, lncRNA FGD5-AS1 acts as a sponge for miR-296-5p. Also, miR-296-5p directly targets STAT3. More importantly, miR-296-5p and STAT3 can affect the function of lncRNA FGD5-AS1 in Dex-treated hBMSCs. Conclusion lncRNA FGD5-AS1 promotes cell proliferation and inhibits apoptosis in steroid-induced ONFH through acting as a sponge for miR-296-5p and upregulation of STAT3.

Construction and Characterizations of Antibacterial Surfaces Based on Self-Assembled Monolayer of Antimicrobial Peptides (Pac-525) Derivatives on Gold

Background: Infection that is related to implanted biomaterials is a serious issue in the clinic. Antimicrobial peptides (AMPs) have been considered as an ideal alternative to traditional antibiotic drugs, for the treatment of infections, while some problems, such as aggregation and protein hydrolysis, are still the dominant concerns that compromise their antimicrobial efficiency in vivo. Methods: In this study, antimicrobial peptides underwent self-assembly on gold substrates, forming good antibacterial surfaces, with stable antibacterial behavior. The antimicrobial ability of AMPs grafted on the surfaces, with or without glycine spaces or a primer layer, was evaluated. Results: Specifically, three Pac-525 derivatives, namely, Ac-CGn-KWRRWVRWI-NH2 (n = 0, 2, or 6) were covalently grafted onto gold substrates via the self-assembling process for inhibiting the growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Furthermore, the alkanethiols HS(CH)10SH were firstly self-assembled into monolayers, as a primer layer (SAM-SH) for the secondary self-assembly of Pac-525 derivatives, to effectively enhance the bactericidal performance of the grafted AMPs. The -(CH)10-S-S-G6Pac derivative was highly effective against S. aureus and E. coli, and reduced the viable amount of E. coli and S. aureus to 0.4% and 33.2%, respectively, after 24 h of contact. In addition, the immobilized AMPs showed good biocompatibility, promoting bone marrow stem cell proliferation. Conclusion: the self-assembled monolayers of the Pac-525 derivatives have great potential as a novel therapeutic method for the treatment of implanted biomaterial infections.

Effects of Transforming Growth Factor-/β 1 and Concentrated Growth Factor on Growth, Proliferation and Osteogenic Differentiation of Bone Marrow Stem Cells

Objective: We aimed to explore the effects of transforming growth factor-β1 (TGF-β1) and concentrated growth factor (CGF) on growth, proliferation and osteogenic differentiation of bone marrow stem cells (BMSCs). Methods: CGF was extracted from rat venous blood. BMSCs was isolated from rats. TGF-β1 or CGF was used to induced BMSCs to observe their effects on BMSCs. The expression levels of BMSCs surface antigens were detected by flow cytometry. ELISA was performed to test the expression of VEGF, TGF-β1, PDGF and SDF-1 in BMSCs. qRT-PCR was carried out to test the expression of COL-I, OPN, OC and ALP in BMSCs. Transwell assay was used to detect the migration rates of BMSCs. The cell morphology of BMSCs was detected by Alizarin red staining. Results: BMSCs were successfully isolated from rats. CGF could significantly increase the expression of TGF-β1, PDGF and VEGF in BMSCs. In addition, TGF-β1 and CGF notably increased the proliferation of BMSCs. And they not only improved the activity of ALD, but also increased the levels of ALP, OPN, COL-I and OCN in BMSCs to prompt the growth of BMSCs. Conclusion: CGF combined with TGF-β1 could prompt the growth, proliferation and osteogenic differentiation of BMSCs.

Induction of apoptosis in retinoid-refractory acute myelogenous leukemia by a novel AHPN analog

AbstractAcute myelogenous leukemia (AML) is a heterogeneous disease consisting of a variety of different leukemic subtypes. While acute promyelocytic leukemia displays marked sensitivity to the differentiating effects of trans-retinoic acid (tRA), other subtypes of AML display resistance. We now describe a novel compound (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC/MM002) that induces apoptosis in the tRA-resistant leukemia cell lines M07e, KG-1, and HL-60R, and in tRA-resistant patient leukemic blasts. The 3-Cl-AHPC totally inhibits leukemia colony formation at concentrations that inhibit committed human bone marrow stem cell proliferation, that is, granulocyte/macrophage colony-forming units (CFU-GMs) by only 30%. Exposure to 3-Cl-AHPC results in caspase activation and the cleavage of poly(adenosine diphosphate) (poly(ADP)) ribose polymerase. While activation of the extracellular signal-regulated kinase (ERK) and p38 pathways is not necessary for 3-Cl-AHPC-mediated apoptosis, maximal apoptosis requires c-Jun N-terminal kinase (JNK) activation. The 3-Cl-AHPC-mediated cleavage of the antiapoptotic B-cell leukemia XL (Bcl-XL) protein to a proapoptotic 18-kDa product is found in both the M07e cell line and patient leukemic blasts. The 3-Cl-AHPC treatment of mice bearing the AML 1498 cell line results in a 3.3-log kill in the leukemic blasts. While 3-Cl-AHPC does not activate retinoic nuclear receptors, it is a potent inducer of apoptosis in AML cells and may represent a novel therapy in the treatment of this disease. (Blood. 2003; 102:3743-3752)

Glucocorticoids decreased Cx43 expression in osteonecrosis of femoral head: The effect on proliferation and osteogenic differentiation of rat BMSCs.

Glucocorticoid (GC)‐induced osteonecrosis of the femoral head (GC‐ONFH) is considered as one of the most serious side effects of long‐term or over‐dose steroid therapy. However, the underlying cause mechanisms are still not fully investigated. We firstly established a rat model of GC‐ONFH and injected lipopolysaccharide (LPS) and methylprednisolone (MPS). We found that the expressions of Cx43, Runx2, ALP and COLⅠ were more decreased than the normal group. Secondly, the isolated rat bone marrow stem cells (BMSCs) were treated with dexamethasone (Dex) in vitro, and the expressions of Cx43, Runx2, ALP and COLⅠ were decreased significantly. Moreover, the results of immunofluorescence staining, alizarin red staining, EdU assay and CCK8 showed that the osteogenic differentiation and the proliferation capacity of BMSCs were decreased after induced by Dex. A plasmid of lentivirus‐mediated Cx43 (Lv‐Cx43) gene overexpression was established to investigate the function of Cx43 in BMSCs under the Dex treatment. Findings demonstrated that the proliferation and osteogenic differentiation abilities were enhanced after Lv‐Cx43 transfected to BMSCs, and these beneficial effects of Lv‐Cx43 were significantly blocked when PD988059 (an inhibitor of ERK1/2) was used. In conclusion, the overexpression of Cx43 could promote the proliferation and osteogenic differentiation of BMSCs via activating the ERK1/2 signalling pathway, which provide a basic evidence for further study on the detailed function of Cx43 in GC‐ONFH.

Adipose-Derived Stem Cells Conditioned Media Promote In Vitro Osteogenic Differentiation of Hypothyroid Mesenchymal Stem Cells

Background: Thyroid hormones have many effects on the physiological functions of cells, including growth, differentiation, and metabolism. Objectives: Recently, studies have shown that the adipose-derived mesenchymal stem cells conditioned medium (ADMSCs-CM) has many osteogenic factors, such as IGF-1, IL-6, and FGFs. Methods: In the current study, mesenchymal stem cells (MSCs) were isolated from two sources; the adipose tissue of the testicular fat pad and the bone marrow of rat, and then characterized by flow cytometry. ADMSCs-CM was collected from the ADMSC in the healthy adult male rats. Hypothyroidism was induced by the administration of the Methimazole during 60 days and confirmed by the analysis of the serum level of T4 and TSH hormones. Cell proliferation and osteogenic differentiation potential of bone marrow stem cells (BMSCs) derived from hypothyroid rats were investigated in the presence and absence of the CM by MTT assay, alkaline phosphatase (ALP) activity, calcium content assay, and bone-related gene expression. Healthy BMSCs were assigned to the control group. Results: Although Cell proliferation was decreased in the hypothyroid BMSCs, there was no significant difference between the control and the hypothyroid-CM groups. Similarly, osteogenic potential was significantly reduced in the hypothyroid group compared to the control and hypothyroid-CM groups according to the ALP, calcium content assays, and gene expression results. There was no significant difference between the hypothyroid-CM group and control. Conclusions: Our results indicated that hypothyroidism can decrease cell proliferation and osteogenic differentiation of BMSCs. Although ADMSCs-CM improved these parameters, it may be a promising candidate for the bone regeneration of the hypothyroidism cases.

Stereocomplex poly(lactic acid)-based composite nanofiber membranes with highly dispersed hydroxyapatite for potential bone tissue engineering

The development of materials with excellent mechanical properties and stem cell affinity for bone repair is a topic that has received significant interest in the field of bone tissue engineering. In this study, stereocomplex poly(lactic acid)-based composite nanofiber membranes that are compatible with bone marrow stem cells (BMSCs) were prepared with poly(d-lactic acid)-grafted hydroxyapatite and enantiomeric poly(lactic acid)s. The mechanical property tests showed that, compared with those from a heated poly(l-lactic acid) (PLLA) nanofiber membrane, the tensile strength and Young's modulus of the most strengthened composite nanofiber membrane herein increased by 30.16% and 34.56%, respectively. The X-ray diffraction results indicated that the stereocomplex crystallite (SC) of poly(lactic acid) formed in heated composite nanofiber membranes. The differential scanning calorimetry results showed that the melting temperature of the SC of heated composite nanofiber membranes was approximately 221 °C. Live/dead staining and scanning electron microscopy were used to observe the proliferation and adhesion of BMSCs cultured on the nanofiber membranes. Compared with the PLLA nanofiber membrane, increased type I collagen expression and improved formation of bone-like nodules can be observed after induction for nine days on the composite nanofiber membrane, indicating the potential application of the composite nanofiber membrane for bone tissue engineering.

The effect of nicotine cessation on human bone marrow stem cell proliferation and differentiation into osteoblasts

The effect of nicotine cessation on human bone marrow stem cell proliferation and differentiation into osteoblasts

Changed cellular functions and aberrantly expressed miRNAs and circRNAs in bone marrow stemcells in osteonecrosis of the femoral head.

The present study aimed to detect the correlations between altered cellular functions in bone marrow stem cells (BMSCs) and osteonecrosis of the femoral head (ONFH). By profiling the aberrant expression of miRNAs and circRNAs in BMSCs isolated from ONFH patients, the present study aimed to further explore the potential regulatory mechanisms of action of circRNAs in ONFH using integrated bioinfor-matics analysis. BMSCs were isolated from seven ONFH patients and seven controls. Cellular functions, including proliferation, apoptosis and differentiation, were compared. miRNA and circRNA sequencing were conducted using RNA samples of three ONFH patients and three controls to identify differentially expressed circRNAs and miRNAs. The expression of hsa_circ_0000219, hsa_circ_0004588 and hsa_circ_0005936 were validated by qPCR. Target miRNAs were also predicted and validated by qPCR and circRNA-miRNA co-expression networks were constructed. BMSCs of ONFH patients displayed decreased proliferation and increased apoptosis during in vitro culturing. In addition, reduced osteogenesis and enhanced adipogenesis were found in the ONFH group. A total of 129 miRNAs and 231 circRNAs were detected to be differentially expressed. The expression levels of hsa_circ_0000219, hsa_circ_0004588 and hsa_circ_0005936 were significantly decreased in BMSCs of ONFH patients. A number of target miRNAs related to cell proliferation, apoptosis and differentiation were predicted for hsa_circ_0000219 and hsa_circ_0005936. The expression levels of miR-144-3p and miR-1270 were found to be elevated in ONFH patients, which was consistent with miRNA sequencing data and competitive endogenous RNA hypothesis. Time-dependent expression patterns of hsa_circ_0000219, hsa_circ_0004588, hsa_circ_0005936, miR-144-3p and miR-1270 were also validated during osteogenic and adipogenic differentiation in BMSCs. The results of the present study substantiated the involvement of BMSCs in ONFH development. hsa_circ_0000219 and hsa_circ_0005936 may regulate the progression of ONFH by mediating the proliferation and differentiation of BMSCs by sponging miRNAs.

Combination of SDF-1 and bFGF promotes bone marrow stem cell-mediated periodontal ligament regeneration.

Stromal cell derived factor-1 (SDF-1) and basic fibroblast growth factor (bFGF) were reported to induce the differentiation of bone marrow stem cells (BMSCs) into cells with characteristics of periodontal ligament fibroblasts. Thus SDF-1 and bFGF may play a positive role in BMSCs-mediated periodontal ligament regeneration. Here, the methylthiazolyldiphenyl tetrazolium bromide (MTT) assay was used to investigate the effect of scaffolds, SDF-1 and bFGF on BMSCs proliferation. RT-PCR and Western blot were used to evaluate gene and protein expression. Beagle dogs were used to establish an animal model of tooth reimplantation and to investigate the effects of scaffolds, BMSCs, SDF-1 and bFGF on periodontal ligament regeneration. X-ray images and micro computed tomography (micro CT) were used to assess morphological changes in replanted teeth and surrounding alveolar bone. H&E staining and Masson’s staining were also performed. BMSCs from Beagle dogs growth on scaffolds consisted of dense structured collagens. SDF-1 and bFGF effectively promoted the differentiation of BMSCs into fibroblasts, periodontal membrane reconstruction, and cell proliferation in vitro. SDF-1 and bFGF also stimulated the expression of type I collagen (Col I), type III collagen (Col III), CXC family chemokine receptor 4 (CXCR4), and S100 calcium binding protein A4 (S100A4), and decreased the expression of alkaline phosphatase (ALP). In our experimental Beagle dog model of tooth extraction and replantation, application of SDF-1 and bFGF significantly elevated periodontal membrane reconstruction and thus supported the survival of replanted teeth. In conclusion, the findings from the present study demonstrated that SDF-1 and bFGF enhance the process of periodontal ligament reconstruction, and provide a basis and reference for the use of stem cell tissue engineering in promoting periodontal membrane regeneration.

S9B-05 SESSION 9B: FACE RESEARCH - PART II INHIBITION OF VASCULARIZED CARTILAGE REGENERATION BY LOADED CURCUMIN SCAFFOLD.

Introduction: Autologous chondrocytes are important cells for cartilage regeneration. But chondrocytes had limited sources. BMSCs are known for their strong proliferative ability and multi-directional differentiation. At present, ectopic ossification of cartilage constructed by BMSCs in subcutaneous environment is an internationally recognized problem. It is generally believed that ectopic ossification is mainly related to vascularization. The key to solve this problem might be vascularization inhibition.Curcumin has anti-angiogenesis effect.The aims of this study were to investigate the regeneration of chondrocytes in the presence of curcumin in-vivo and clarify how to regulate curcumin loading materials to inhibit angiogenesis without affecting the BMSCs proliferation. Methods: The curcumin loaded materials were set 2groups (serum-free medium and PBS) and treated at 3 different time-point stages(24h,48h,72h).For the PBS group, the absorbance rate was measured at the beginning of each stage to detect the curcumin concentration standard line.For the serum-free medium group, the loaded materials were given to vascular endothelial cells and BMSCs at the beginning of each stage.The tests for concentrations of curcumin solution, cell scratch test, cell cycle, and apoptosis tests were carried.The curcumin loaded materials were implanted subcutaneously into nude mice. After 3,6,12weeks tests HE,S/Ostaining, CD31 immunohistochemical, CD31 immunofluorescence were performed. Seed cells from rabbit ear cartilage were cultured 4weeks in vitro and 3,6weeks in vivo.Constructs were implanted subcutaneously into the nude mice. Results: The results of CCK8 showed that the levels of curcumin released after the first 24 hours were higher than those after 48 and 72 hours. However, the effects on BMSCs growth were not obvious. The cell scratch test showed that the apoptosis of vascular endothelial cells began at the curcumin concentration of 30um while BMSCs apoptosis began at 40um. Therefore, the curcumin concentration of 30um can be considered as appropriate since it can inhibit the growth of vascular endothelial cells without affecting the proliferation of BMSCs. The results of gross view images, CD31 immunohistochemistry and immunofluorescence showed that scaffolds loaded with curcumin could significantly inhibit the vascularization (compared to the control group). After 4 weeks of in-vitro and 3,6weeks of in-vivo, both constructs were able to form cartilaginous tissues. Conclusion: With the appropriate concentration, curcumin loaded scaffolds can inhibit the vascular endothelial cells without any obvious effects on bone marrow stem cells proliferation. Therefore, they could be successfully used in the construction and tissue engineering of cartilages in subcutaneous animal models. That gives curcumin loaded scaffolds a great potential application prospect.

The role of lncRNA RP11-154D6 in steroid-induced osteonecrosis of the femoral head through BMSC regulation.

Bone marrow stem cells (BMSCs) are involved in steroid-induced osteonecrosis of the femoral head (ONFH). Long noncoding RNAs (lncRNAs) have been demonstrated to regulate functions of BMSCs, especially their differentiation, and have been reported to be implicated in diverse bone disorders. However, the roles of lncRNAs in the progression of steroid-induced ONFH remain unknown. BMSCs were isolated from healthy controls or patients with steroid-induced ONFH. RNA-sequencing was used to identify differentially expressed (DE) coding and noncoding transcripts and a lncRNA-messenger RNA coexpression network were created. The function of lncRNA RP11-154D6 in the proliferation, apoptosis, and differentiation of BMSCs was identified by overexpressing or silencing its expression. Altogether, 3114 DE messenger RNAs and 572 DE lncRNAs were identified and the expression of lncRNA RP11-154D6 was significantly decreased in BMSCs of patients with ONFH. Although no effects on proliferation and apoptosis were identified, we proved lncRNA RP11-154D6 promoted osteogenic differentiation while inhibiting adipogenic differentiation in BMSCs. Aberrant expression of lncRNA was identified in BMSCs of steroid-induced patients with ONFH. Our results demonstrated that lncRNA RP11-154D6 might contribute to the progression of steroid-induced ONFH through regulating the behavior of BMSCs and might provide new insights into the pathogenesis as well as treatment for steroid-induced ONFH.

Rational design of gelatin/nanohydroxyapatite cryogel scaffolds for bone regeneration by introducing chemical and physical cues to enhance osteogenesis of bone marrow mesenchymal stem cells.

Identification of key components in the chemical and physical milieu for directing osteogenesis is a requirement in the investigation of tissue engineering scaffolds for advancement of bone regeneration. In this study, we engineered different gelatin-based cryogels and studied the effect of nanohydroxyapatite (nHAP) and crosslinking agents on scaffold properties and its osteogenic response towards bone marrow stem cells (BMSCs). The cryogels examined are 5% gelatin and 5% gelatin/2.5% nHAP, crosslinked either with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or glutaraldehyde (GA). We confirmed that nHAP or the crosslinking agent has no effects on scaffold pore size and porosity. Nonetheless, incorporation of nHAP increased mechanical strength, swelling ratio and degree of crosslinking, but decreased degradation rate. Cryogels crosslinked with EDC showed faster degradation and promoted osteogenic differentiation of BMSCs while those prepared from GA crosslinking promoted proliferation of BMSCs. Furthermore, osteogenic differentiation was always enhanced in the presence of nHAP irrespective of the culture medium (normal or osteogenic) used but osteogenic medium always provide a higher extent of osteogenic differentiation. Employing gelatin/nHAP cryogel crosslinked by EDC in a bioreactor for dynamic culture of BMSCs, cyclic compressive mechanical simulation was found to be beneficial for both cell proliferation and osteogenic differentiation. However, the optimum conditions for osteogenic differentiation and cell proliferation were found at 30% and 60% strain, respectively. We thus demonstrated that osteogenic differentiation of BMSCs could be tuned by taking advantages of chemical cues generated from scaffold chemistry or physical cues generated from dynamic cell culture in vitro. Furthermore, by combining the best cryogel preparation and in vitro cell culture condition for osteogenesis, we successfully employed in vitro cultured cryogel/BMSCs constructs for repair of rabbit critical-sized cranial bone defects.

MTORC1 signaling is essential for neurofibromatosis type I gene modulated osteogenic differentiation of BMSCs.

Neurofibromatosis type I (NF1), which is caused by mutations in the NF1 gene, is a common autosomal dominant genetic disease leading to skeletal abnormalities. Both NF1 gene and mammalian target of rapamycin complex 1 (mTORC1) signaling are associated with the osteogenic differentiation of bone marrow stem cells (BMSCs). In this study, we hypothesized that mTORC1 signaling is involved in NF1-modulated osteoblast differentiation of BMSCs. Human BMSCs were cultured in an osteogenic induction medium. The expression of NF1 was either inhibited or overexpressed by transfecting NF1 with a specific small interfering RNA (siRNA) or pcDNA3.0 plasmid, respectively. In addition, an mTORC1 signaling inhibitor and agonist were used to investigate the effects of mTORC1 on NF1-modulated osteogenic differentiation of BMSCs. The results indicated that inhibiting the expression of NF1 with siRNA significantly decreased the mRNA levels of NF1, whereas overexpressing the expression of NF1 with pcDNA3.0 plasmid significantly increased the mRNA levels of NF1 at days 3, 7, 14 and 21 after culture. We observed reduced osteogenic differentiation and cell proliferation in the NF1-siRNA group and enhanced osteogenic differentiation and cell proliferation of BMSCs in the NF1-pcDNA3.0 group. The activity of mTORC1 signaling (p-mTORC1, p-S6K1, and p-4EBP1) was significantly upregulated in the NF1-siRNA group and significantly inhibited in the NF1-pcDNA3.0 group, 7 and 14 days after culture. The effects of NF1-siRNA and NF1-pcDNA3.0 on osteogenic differentiation of BMSCs and cell proliferation were reversed by mTORC1 inhibitor and agonist, respectively. In conclusion, NF1 modulates osteogenic differentiation and cell proliferation of human BMSCs and mTORC1 signaling is essential for this process.

Hybrid scaffolds of Mg alloy mesh reinforced polymer/extracellular matrix composite for critical-sized calvarial defect reconstruction.

The challenge of developing scaffolds to reconstruct critical-sized calvarial defects without the addition of high levels of exogenous growth factor remains relevant. Both osteogenic regenerative efficacy and suitable mechanical properties for the temporary scaffold system are of importance. In this study, a Mg alloy mesh reinforced polymer/demineralized bone matrix (DBM) hybrid scaffold was designed where the hybrid scaffold was fabricated by a concurrent electrospinning/electrospraying of poly(lactic-co-glycolic acid) (PLGA) polymer and DBM suspended in hyaluronic acid (HA). The Mg alloy mesh significantly increased the flexural strength and modulus of PLGA/DBM hybrid scaffold. In vitro results demonstrated that the Mg alloy mesh reinforced PLGA/DBM hybrid scaffold (Mg-PLGA@HA&DBM) exhibited a stronger ability to promote the proliferation of bone marrow stem cells (BMSCs) and induce BMSC osteogenic differentiation compared with control scaffolding materials lacking critical components. In vivo osteogenesis studies were performed in a rat critical-sized calvarial defect model and incorporated a variety of histological stains and immunohistochemical staining of osteocalcin. At 12weeks, the rat model data showed that the degree of bone repair for the Mg-PLGA@HA&DBM scaffold was significantly greater than for those scaffolds lacking one or more of the principal components. Although complete defect filling was not achieved, the improved mechanical properties, promotion of BMSC proliferation and induction of BMSC osteogenic differentiation, and improved promotion of bone repair in the rat critical-sized calvarial defect model make Mg alloy mesh reinforced PLGA/DBM hybrid scaffold an attractive option for the repair of critical-sized bone defects where the addition of exogenous isolated growth factors is not employed.