Oct4 facilitates chondrogenic differentiation of mesenchymal stem cells by mediating CIP2A expression.

Bone development and cartilage formation require strict modulation of gene expression for mesenchymal stem cells (MSCs) to progress through their differentiation stages. Octamer-binding transcription factor 4 (Oct4) expression is generally restricted to developing embryonic pluripotent cells, but its role in chondrogenic differentiation (CD) of MSCs remains unclear. We therefore investigated the role of Oct4 in CD using a microarray, quantitative real-time polymerase chain reaction, and western blotting. The expression of Oct4 was elevated when the CD of cultured MSCs was induced. Silencing Oct4 damaged MSC growth and proliferation and decreased CD, indicated by decreased cartilage matrix formation and the expression of Col2a1, Col10a1, Acan, and Sox9. We found a positive correlation between the expression of CIP2A, a natural inhibitor of protein phosphatase 2A (PP2A) and that of Oct4. Cellular inhibitor of PP2A (CIP2A) expression gradually increased after CD. Overexpression of CIP2A in MSCs with Oct4 depletion promoted cartilage matrix deposition as well as Col2a1, Col10a1, Acan, and Sox9 expression. The chondrogenic induction triggered c-Myc, Akt, ERK, and MEK phosphorylation and upregulated c-Myc and mTOR expression, which was downregulated upon Oct4 knockdown and restored by CIP2A overexpression. These findings indicated that Oct4 functions as an essential chondrogenesis regulator, partly via the CIP2A/PP2A pathway.

LINC02288 promotes chondrocyte apoptosis and inflammation through miR-374a-3p targeting RTN3.

BACKGROUND: Dysregulation of long non-coding RNAs (lncRNAs) is related to the occurrence of osteoarthritis (OA). In the present study, we explored the role of LINC02288 and its regulatory mechanism in OA development. METHODS: GSE113825 was obtained from Gene Expression Omnibus (GEO) database and analyzed to identify the differentially expressed lncRNAs in OA. Gene enrichment analyses and Kyoto Encyclopedia of Genes and Genomes biological process analysis were performed through Metascape (http://metascape.org/gp). The interactions among LINC02288, miR-374a-3p and RTN3 were determined using RNA immunoprecipitation (RIP) assays and dual luciferase reporter assays. Chondrocyte apoptosis was examined using flow cytometry. Western blot assays were conducted to assess the pro-apoptotic and anti-apoptotic markers. RESULTS: We identified a total of 4,491 differentially expressed lncRNAs. We focused on LINC02288 as the top-ranked up-regulated lncRNA in OA as indicated by a significant p-value. LINC02288 was significantly up-regulated, which was further verified by a real-time polymerase chain reaction. Down-regulation of LINC02288 significantly reduced the apoptosis of OA chondrocytes induced by interleukin-1ß and the production of pro-inflammatory cytokines. These effects were further verified in an OA rat model. An RIP assay and dual luciferase assay further confirmed that LINC02288 served as a sponge of miR-374a-3p. Moreover, the overexpression of RTN3 could partially reverse the effects of LINC02288 knockdown, mediating inhibitory effects on chondrocyte apoptosis and the inflammatory response. Down-regulation of LINC02288 alleviated OA development in an in vivo OA animal model. CONCLUSIONS: Our findings indicate that LINC02288 contributes to OA progression by targeting the miR-374a-3p/RTN3 axis, which might provide a promising molecular therapy strategy for OA.

CircADAMTS6/miR-431-5p axis regulate interleukin-1ß induced chondrocyte apoptosis.

BACKGROUND: Growing evidence suggests that circular RNAs (circRNAs) are involved in the development of osteoarthritis (OA). The present study aimed to explore the CircADAMTS6/miR-431-5p axis with respect to regulating interleukin-1ß (IL-1ß) induced chondrocyte apoptosis. METHODS: We first evaluated the differentially expressed circRNAs between normal chondrocytes and interleukin (IL)-1ß-stimulated chondrocytes. Then, bioinformatic analysis was performed to identify the role and function of circADAMTS6. Small interfering RNA-expressing or overexpressing circADAMTS6 lentiviral vectors were used for transduction of chondrocytes. Annexin-V-fluorescein isothiocyanate (FITC) double staining was performed to measure the apoptotic rate of the chondrocytes in each group. Finally, a dual luciferase reporter assay was performed to identify the target relationship between circADAMTS6 and miR-431-5p. RESULTS: After treatment with IL-1ß, circADAMTS6 was down-regulated compared to the normal chondrocyte group. The overexpression of circADAMTS6 inhibited apoptosis in human chondrocytes, as indicated by annexin-V-FITC double staining. However, overexpression of miR-431-5p had the opposite effect. A dual luciferase reporter assay indicated that circADAMTS6 could directly binding with miR-431-5p. CONCLUSIONS: Our findings demonstrate that the circADAMTS6/miR-431-5p axis comprises a new target for OA. Bioinformatic analysis suggested that circADAMTS6 acted as a sponge of miR-431-5p.

Is soft tissue repair a right choice to avoid early dislocation after THA in posterior approach?

BACKGROUND: Dislocation is the second most common complication after total hip arthroplasty (THA). The effectiveness of soft tissue repair to reduce dislocation rate is still debated and thus a meta-analysis was conducted. METHODS: A systematic search in PubMed, Embase, and Cochrane databases was conducted for this meta-analysis. INCLUSION CRITERIA: clinical comparative trials on the use of soft tissue repair including rotators and capsule repair in primary THA. The main data outcome were the incidences of early hip dislocation after primary THA. HSS score, incidence of other complications was also included in the outcomes. RESULTS: A total of 4816 cases were included for the analysis from ten studies (3 RCTs/7 Retrospective trials). Overall, the soft tissue repair group showed a significant lower early dislocation rate and higher HSS score compared to the no repair group; but no significant difference was observed between the two groups in regards to the early dislocation rate in RCT studies only. The capsule repair group showed a significant lower early dislocation rate than no capsule repair group while no significant difference was observed between the rotators repair group and no rotators repair group. In all included studies, 4 greater trochanter fractures, 2 sciatic nerve palsies and 1 infection were reported in soft tissue repair group while no cases were observed in the no repair group. CONCLUSIONS: The efficacy of soft tissue repair is positive but still not conclusive to reduce the early dislocation rate after primary THA while soft tissue repair may bring more other complications. Capsule repair seems more effective than rotators repair only.

Clinical Efficacy of Intra-Articular Injection with P-PRP Versus that of L-PRP in Treating Knee Cartilage Lesion: A Randomized Controlled Trial.

OBJECTIVE: Platelet-rich plasma（PRP), with different concentration of leukocytes, may lead to varying effects in the treatment of cartilage lesions. So far, current research has not shown enough evidence on this. To evaluate the clinical efficacy and safety of intra-articular injection with pure platelet-rich plasma (P-PRP) versus those of leukocyte platelet-rich plasma (L-PRP) in treating knee cartilage lesions, we conducted a double-blind, randomized controlled clinical trial with a larger sample and longer follow-up period. METHODS: From October 2019 to October 2020, 95 patients were invited to participate in our study, and 60 (63.2%) were randomized to P-PRP (n = 30) or L-PRP (n = 30) groups. Patients from the two groups were treated with knee intra-articular injections of P-PRP or L-PRP. Visual analog scale (VAS) and Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores were assessed using an unpaired t-test for independent samples preoperatively and at 6 weeks, 12 weeks, 6 months, and 12 months after intervention. RESULTS: We followed up 27 cases in the P-PRP group and 26 cases in the L-PRP group. No significant differences in VAS and WOMAC scores were found between the two groups before the intervention (p > 0.05). The WOMAC Pain and VAS-Motions scores of the P-PRP group were significantly lower than those of the L-PRP group at 6 weeks after the intervention (p < 0.05). While the long-term clinical efficacy of both injections was similar and weakened after 12 months, more adverse events were found in the L-PRP group. CONCLUSIONS: The short-term results demonstrate a positive effect in reducing pain and improving function in patients with knee cartilage lesions in the two groups. While the P-PRP injection showed better clinical efficacy in the early phase of postoperative rehabilitation and resulted in fewer adverse events, long-term follow-up showed similar and weakened efficacy after 12 months. TRIAL REGISTRATION: ChiCTR1900026365. Registered on October 3, 2019, http://www.chictr.org.cn/showproj.aspx?proj=43911.

A high throughput bispecific antibody discovery pipeline.

Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy, but inefficiency in the current discovery has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model, we demonstrate that our single-cell platform possesses a high throughput screening efficiency of up to one and a half million variant library cells per run and can isolate rare functional clones at a low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones, including extremely rare ones (~ 0.001% abundance). We also discovered BiTEs that exhibit novel properties and insights to design variable preferences for functionality. We expect our single-cell platform to not only increase the discovery efficiency of new immunotherapeutics, but also enable identifying generalizable design principles based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

An alternative method for personalized tourniquet pressure in total knee arthroplasty: a prospective randomized and controlled study.

Tourniquet use always carries potential risks, which can range from mild transient functional impairments of thigh pain, skin blisters to severe permanent dysfunction of limb paralysis, nerve injuries or compartment syndrome. The ideal method for minimizing intraoperative tourniquet pressure (TP) for reducing postoperative complications remains controversial. In this prospective, randomized and controlled study, we reinvestigated an estimation formula for TP based on thigh circumferences and systolic blood pressure (SBP) with two traditional methods for TP determination in total knee arthroplasty (TKA): SBP plus 100 mmHg and a fixed value of 300 mmHg. TP values and postoperative thigh pain scores were compared among three groups. The intraoperative TP value of the formula-calculated group was lower than that of the traditional groups (14.7 mmHg, P = 0.3475 and 94.7 mmHg, P < 0.0001, respectively), while no differences of hemostatic effect at the surgical fields and wound complications were detected among groups. The thigh pain scores at the tourniquet site decreased gradually over time and the estimation group had the lowest scores at each timepoint after surgery. Estimation method for TP was easy and rapid, without relying on specific equipment. It could provide a practical low TP and comparable hemostatic effect in TKA using an inflating tourniquet.

Culture of Mesenchymal Stem Cells Derived From the Infrapatellar Fat Pad Without Enzyme and Preliminary Study on the Repair of Articular Cartilage Defects in Rabbits.

Objective: The aim of the study was to evaluate the advantages of without enzyme isolating patellar fat pad-derived mesenchymal stem cells (IPFP-SCs) and the feasibility of cartilage repair. Methods: The IPFP-SCs were isolated using the without enzyme method and compared with the IPFP-SCs obtained by the traditional enzyme digestion method in terms of cell proliferation ability, characterization, and differentiation ability, and the differences in chondrogenic induction and differentiation between the two groups were compared. Twenty-four New Zealand rabbits were randomly divided into four groups (n = 6). After the articular cartilage defects were modeled, different preparations were injected into the joint cavity. The rabbits in the group A were injected with the mixture of IPFP-SCs and pure PRP (P-PRP), separated using the without enzyme method, while those in the group B were injected with the mixture of IPFP-SCs and P-PRP separated with the digestion method, while those in the group C were injected with SVF separated using the without enzyme method, and those in the group D were injected with normal saline. At 6 weeks and 12 weeks after operation, the cartilage repair of rabbit joint specimens was observed and evaluated by gross observation and histological staining, and the effects of different IPFP-SCs application forms in repairing cartilage defects were compared. Results: The time required to obtain IPFP-SCs by enzyme-free isolation was significantly less than that by enzyme digestion, while the acquisition rate of primary cells was significantly lower than that by enzyme digestion. After culture and amplification, the two IPFP-SCs from different sources did not show significant differences in cell proliferation, cell phenotype, and differentiation ability. In animal experiments, groups A and B had the best effect on the repair of cartilage defects, and there was no significant difference between the two groups. The repair effect in group C was weaker than that in the former two groups, but it was relatively better than that in group D. Conclusion: It is more time-saving to obtain IPFP-SCs by the without enzyme method than by enzymatic digestion, and there is no significant difference in cell identification and differentiation potential between the two methods. However, the rate of obtaining primary cells was significantly lower than that with the enzyme digestion method. IPFP-SCs showed good repair effect in the rabbit animal cartilage defect model, providing ideas and reference for the clinical application of stem cells in repairing articular cartilage.

Regulating effect of Circ_ATRNL1 on the promotion of SOX9 expression to promote chondrogenic differentiation of hAMSCs mediated by MiR-145-5p.

Circ_ATRNL1 is significantly highly expressed in cartilage tissues of patients with osteoarthritis. This study explored the role and mechanism of circ_ATRNL1 in cartilage differentiation of human adipose-derived mesenchymal stem cells (hAMSCs). hAMSCs were isolated and identified by flow cytometry. The degree of chondrocyte and adipogenic differentiation was assessed using Alcian blue staining and Oil Red O staining, respectively. The expressions of cartilage- and adipogenic-related genes, circ_ATRNL1, and SOX9 were detected by reverse transcription quantitative polymerase chain reaction. The correlation between SOX9 and circ_ATRNL1 was analyzed using Pearson test. Bioinformatics and luciferase analysis were used to detect the overlapped target miRNAs of circ_ATRNL1 and SOX9. The role of circ_ATRNL1/miRNA/SOX9 was examined using functional rescue assays. hAMSCs were identified as CD90-, CD105-, and CD44-positive. The degree of cartilage differentiation of hAMSCs was significantly enhanced after 2 weeks. Cartilage-related genes, circ_ATRNL1 and SOX9, were significantly upregulated, and positively correlated with each other. Circ_ATRNL1 overexpression enhanced hAMSC proliferation and differentiation into chondrogenesis, and promoted the expressions of COL2, Aggrecan, and SOX9. Overexpression of circ_ATRNL1 inhibited the adipogenic differentiation of hAMSCs and the expressions of adipogenic-related genes. miR-145-5p was a target miRNA for circ_ATRNL1 and SOX9. miR-145-5p mimic inhibited hAMSC differentiation toward cartilage, and inhibited the expression of cartilage-related factors. miR-145-5p mimic effectively reversed the regulating effect of circ_ATRNL1 on hAMSCs. Circ_ATRNL1 regulates the promotion of SOX9 expression to promote chondrogenic differentiation of hAMSCs mediated by miR-145-5p.

Over-expression of MEG3 promotes differentiation of bone marrow mesenchymal stem cells into chondrocytes by regulating miR-129-5p/RUNX1 axis.

This study explored the role of MEG3 in the cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs). We investigated the effects of over-expression and knockdown of MEG3 on cell viability, cell differentiation, and the expressions of MEG3, miR-129-5p, COL2, chondrocyte differentiation-related genes (sry-type high-mobility-group box 9 (SOX9), SOX5, Aggrecan, silent information regulator 1 (SIRT1), and Cartilage oligomeric matrix protein (COMP)). The targeting relationship between MEG3 and miR-129-5p and the target gene of miR-129-5p was confirmed through Starbase, TargetScan and luciferase experiments. Finally, a series of rescue experiments were conducted to study the regulatory effects of MEG3 and miR-129-5p. BMSCs were identified as CD29+ and CD44+ positive, and their differentiation was time-dependent. As BMSCs differentiated, MEG3 expression was up-regulated, but miR-129-5p was down-regulated. Over-expressed MEG3 promoted the viability and differentiation of BMSCs, up-regulated the expressions of COL2 and chondrocyte differentiation-related genes, and inhibited miR-129-5p. Runt-related transcription factor 1 (RUNX1) was negatively regulated as a target gene of miR-129-5p. Results of rescue experiments showed that the inhibitory effect of miR-129-5p mimic on BMSCs could be partially reversed by MEG3. Over-expression of MEG3 regulated the miR-129-5p/RUNX1 axis to promote the differentiation of BMSCs into chondrocytes. This study provides a reliable basis for the application of lncRNA in articular cartilage injury.

Synergic adhesive chemistry-based fabrication of BMP-2 immobilized silk fibroin hydrogel functionalized with hybrid nanomaterial to augment osteogenic differentiation of rBMSCs for bone defect repair.

Bone defect repair and tissue engineering is specifically challenging process because of the distinctive morphological and structural behaviours of natural bone with complex healing and biochemical mechanisms. In the present investigation, we designed dopamine adhesive chemistry-based fabrication of silk fibroin hydrogel (SFD) with incorporation of nano-hydroxyapatite (nHA)-graphene oxide (GO) hybrid nanofillers with well-arranged porous morphology immobilized with bone morphogenic protein-2 (BMP-2) for the effective in vitro rabbit bone marrow derived mesenchymal stem cells loading compatibility and in vivo new bone regrowth and collagen deposition ability. We have achieved bone-specific hydrogel scaffolds with upgraded structural features, mechanical properties and particularly promoted in vitro osteogenic differentiation and compatibility of rabbit bone marrow mesenchymal stem cells (rBMSCs). Structural and microscopic analyses established greater distributions of components and well-ordered and aligned porous structure of the hydrogel network. In vivo result of new bone regrowth was promisingly higher in the Bm@nHG-SFD hydrogel (85%) group as compared to the other treatment groups of nHG-SFD (77%) and nH-SFD (64%) hydrogel. Overall, we summarized that morphologically improved hydrogel material with immobilization of BMP-2 could be have more attentions for new generation bone regeneration therapies.

The clinical efficacy of arthroscopic therapy with knee infrapatellar fat pad cell concentrates in treating knee cartilage lesion: a prospective, randomized, and controlled study.

INTRODUCTION: To evaluate the clinical efficacy of arthroscopic therapy with infrapatellar fat pad cell concentrates in treating knee cartilage lesions, we conducted a prospective randomized single-blind clinical study of controlled method. METHODS: Sixty cases from Shanghai Changzheng Hospital from April 2018 to December 2019 were chosen and randomly divided into 2 groups equally. Patients in the experiment group were treated through knee arthroscopy with knee infrapatellar fat pad cell concentrates containing mesenchymal stromal cells, while patients in the control group were treated through regular knee arthroscopic therapy. VAS and WOMAC scores were assessed at pre-operation, and 6 weeks, 12 weeks, 6 months, and 12 months after intervention. MORCART scores were assessed at pre-operation and 12 months after intervention. RESULTS: Twenty-nine cases in the experiment group and 28 cases in the control group were followed up. No significant difference in VAS, WOMAC, and MOCART scores were found between the two groups before surgery (P > 0.05). The WOMAC total and WOMAC function scores of the experiment group were significantly lower than those of the control group 6 months and 12 months after surgery (P < 0.05). The VAS rest and VAS motion scores of the experiment group were found significantly lower than those of the control group 12 months after surgery (P < 0.05). The MOCART scores of the experiment group were found significantly higher compared with the control group 12 months after surgery (P < 0.05). No significant difference in WOMAC stiffness scores were found between the two groups. CONCLUSIONS: The short-term results of our study are encouraging and demonstrate that knee arthroscopy with infrapatellar fat pad cell concentrates containing mesenchymal stromal cells is safe and provides assistance in reducing pain and improving function in patients with knee cartilage lesions. TRIAL REGISTRATION: ChiCTR1800015379. Registered on 27 March 2018, http://www.chictr.org.cn/showproj.aspx?proj=25901 .

Decellularized extracellular matrix loaded with IPFP-SC for repairing rabbit osteochondral defects.

BACKGROUND: Tissue engineering is widely applied to treat osteochondral damage in osteoarthritis (OA). However, the superposition of seed cells, material scaffolds, inducing factors, and microenvironmental factors limit their practical application. We intended to develop a novel tissue engineering method for improving the repairment of osteochondral damage and to discuss its effect on repairing osteochondral defects. METHODS: The combined decellularization methods of physics, chemistry and enzymes were used to decellularize rabbit rib cartilage and articular cartilage, and rabbit decellularizated osteochondral composite scaffolds were prepared. The structure and organization of the scaffolds were analyzed. We extracted and identified infrapatellar fat pad stem cells (IPFP-SCs) from healthy rabbits and OA rabbit, which were different in viability, migration, osteogenic and chondrogenic differentiation. Finally, a variety of decellularizated bone cartilage composite scaffolds were loaded with rabbit IPFP-SC for in vitro and in vivo studies. RESULTS: The decellularization effect was strong, and the organic ingredients were lost. The layered scaffold showed lower density, greater porosity, larger pore size and water absorption than the whole scaffold, but the mechanical properties of the two scaffolds were low. IPFP-SCs were successfully extracted, and the migration and cartilage ability of IPFP-SCs in OA group were weak. The decellularized scaffold showed a high biocompatibility. The structure and composition of osteochondral promoted osteogenic differentiation and chondrogenic differentiation of IPFP-SCs. Moreover, the decellularized extracellular matrix loaded with IPFP-SC had the strongest repairing effect. CONCLUSION: The decellularized extracellular matrix loaded with IPFP-SC showed a better repair effect on rabbit osteochondral defects.

Fibrin Glue-Kartogenin Complex Promotes the Regeneration of the Tendon-Bone Interface in Rotator Cuff Injury.

OBJECTIVE: Rotator cuff injury healing is problematic because the tendon-bone junction often forms cicatricial tissues, rather than fibrocartilage, which leads to mechanical impairment and is prone to redamage. Kartogenin (KGN) is a newly discovered small molecule compound which can induce cartilage formation through chondrogenesis of endogenous mesenchymal stem cells. METHODS: In this study, we used KGN with fibrin glue (FG) to repair the rotator cuff injury by promoting the formation of fibrocartilage at the tendon to bone interface. Firstly, we assessed the release rate of KGN from the FG-KGN complex and then created a rabbit rotator cuff tendon graft-bone tunnel model. The rabbits received saline, FG-KGN, or FG injections onto the tendon to bone interface after injury. Shoulder tissues were harvested at 6 and 12 weeks, and the sections were stained with HE and Safranin O/Fast green. The samples were assessed by histologic evaluation and biomechanical testing. Synovial mesenchymal stem cells derived from the synovial tissue around the rotator cuff were harvested for western blotting and qRT-PCR analysis. RESULTS: KGN was released rapidly from the FG-KGN complex during first 4 hrs and followed by a slow release until 7 days. The tendon graft-bone interface in the control (saline) group and the FG group was filled with scar tissue, rather than cartilage-like tissue, and only a small number of chondrocytes were found at the adjacent bone surface. In the FG-KGN group, the tendon to bone interface was fully integrated and populated by chondrocytes with proteoglycan deposition, indicating the formation of fibrocartilage-like tissues. At 12 weeks, the maximum tensile strength of the FG-KGN group was significantly higher than that of the FG and control groups (P < 0.01). The RNA expression levels of tendinous genes such as Tenascin C and the chondrogenic gene Sox-9 were substantially elevated in SMSCs treated with the FG-KGN complex compared to the other two groups. CONCLUSION: These results indicated that fibrin glue is an effective carrier for KGN, allowing for the sustained release of KGN. The FG-KGN complex could effectively promote the regeneration and formation of fibrocartilage tissue of the tendon-bone interface in the rabbit rotator cuff tendon graft-bone tunnel model.

Exosomes from Kartogenin-Pretreated Infrapatellar Fat Pad Mesenchymal Stem Cells Enhance Chondrocyte Anabolism and Articular Cartilage Regeneration.

OBJECTIVE: To evaluate the effect of Kartogenin-pretreated exosomes derived from infrapatellar fat pad mesenchymal stem cells on chondrocyte in vitro and articular cartilage regeneration in vivo. METHODS: Infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) were isolated from rabbits to harvest exosomes. After identification of mesenchymal stem cells and exosomes, rabbit chondrocytes were divided into three groups for further treatment: the EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells), KGN-EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN), and control group. After processing and proliferation, phenotypic changes of chondrocytes were measured. In the in vivo study, 4 groups of rabbits with articular cartilage injury were treated with KGN-EXO, EXO, IPFP-MSCs, and control. Macroscopic evaluation and histological evaluation were made to figure out the different effects of the 4 groups on cartilage regeneration in vivo. RESULTS: The proliferation rate of chondrocytes in the EXO or KGN-EXO group was significantly higher than that in the control group (P < 0.05). The qRT-PCR results showed that the expression of Sox-9, Aggrecan, and Col II was the highest in the KGN-EXO group compared with the EXO group and the control group (P < 0.05). The results of Western blot were consistent with the results of qRT-PCR. In vivo, the cartilage defects in the KGN-EXO group showed better gross appearance and improved histological score than those in IPFP-MSC groups, EXO groups, and control groups (P < 0.05). At 12 weeks, the defect site in the KGN-EXO group was almost completely repaired with a flat and smooth surface, while a large amount of hyaline cartilage-like structures and no obvious cracks were observed. CONCLUSION: Our study demonstrates that the exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN have potent ability to induce chondrogenic differentiation of stem cells, effectively promoting the proliferation and the expression of chondrogenic proteins and genes of chondrocytes. The KGN-EXO can also promote the repair of articular cartilage defects more effectively, which can be used as a potential therapeutic method in the future.

Comparison between Intra-Articular Injection of Infrapatellar Fat Pad (IPFP) Cell Concentrates and IPFP-Mesenchymal Stem Cells (MSCs) for Cartilage Defect Repair of the Knee Joint in Rabbits.

Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic method in regenerative medicine. Our previous research adopted a simple nonenzymatic strategy for the preparation of a new type of ready-to-use infrapatellar fat pad (IPFP) cell concentrates. The aim of this study was to compare the therapeutic efficacy of intra-articular (IA) injection of autologous IPFP cell concentrates and allogeneic IPFP-MSCs obtained from these concentrates in a rabbit articular cartilage defect model. IPFP-MSCs sprouting from the IPFP cell concentrates were characterized via flow cytometry as well as based on their potential for differentiation into adipocytes, osteoblasts, and chondrocytes. In the rabbit model, cartilage defects were created on the trochlear groove, followed by treatment with IPFP cell concentrates, IPFP-MSCs, or normal saline IA injection. Distal femur samples were evaluated at 6 and 12 weeks posttreatment via macroscopic observation and histological assessment based on the International Cartilage Repair Society (ICRS) macroscopic scoring system as well as the ICRS visual histological assessment scale. The macroscopic score and histological score were significantly higher in the IPFP-MSC group compared to the IPFP cell concentrate group at 12 weeks. Further, both treatment groups had higher scores compared to the normal saline group. In comparison to the latter, the groups treated with IPFP-MSCs and IPFP cell concentrates showed considerably better cartilage regeneration. Overall, IPFP-MSCs represent an effective therapeutic strategy for stimulating articular cartilage regeneration. Further, due to the simple, cost-effective, nonenzymatic, and safe preparation process, IPFP cell concentrates may represent an effective alternative to stem cell-based therapy in the clinic.

The mineralization, drug release and in vivo bone defect repair properties of calcium phosphates/PLA modified tantalum scaffolds.

Calcium phosphate based biomaterials have been widely studied in biomedical areas. Herein, amorphous calcium phosphate (ACP) nanospheres and hydroxyapatite (HA) nanorods were separately prepared and used for coating tantalum (Ta) scaffolds with a polymer of polylactide (PLA). We have found that different crystal phases of calcium phosphate coated on Ta scaffolds displayed different effects on the surface morphologies, mineralization and bovine serum albumin (BSA) release. The ACP-PLA and HA-PLA coated on Ta scaffold were more favorable for in vitro mineralization than bare and PLA coated Ta scaffolds, and resulted in a highly hydrophilic surfaces. Meanwhile, the osteoblast-like cells (MG63) showed favorable properties of adhesion and spreading on both ACP-PLA and HA-PLA coated Ta scaffolds. The ACP-PLA and HA-PLA coated Ta scaffolds showed a high biocompatibility and potential applications for in vivo bone defect repair.

Simplified Chinese version of the Forgotten Joint Score (FJS) for patients who underwent joint arthroplasty: cross-cultural adaptation and validation.

BACKGROUND: The Forgotten Joint Score (FJS) is a newly developed health-related quality of life (HRQoL) questionnaire designed to evaluate the awareness after total knee arthroplasty (TKA). This study cross-culturally adapted and psychometrically validated a simplified Chinese version of the FJS (SC-FJS). METHODS: Cross-cultural adaptation was performed according to the internationally recognized guidelines. One-hundred and fifty participants who underwent primary TKA were recruited in this study. Cronbach's α and intra-class correlations were used to determine reliability. Construct validity was analyzed by evaluating the correlations between SC-FJS and the Knee Injury and Osteoarthritis Outcome Score (KOOS) and the short form (36) health survey (SF-36). RESULTS: Each of the 12 items was properly responded and correlated with the total items. SC-FJS had excellent reliability [Cronbach's α = 0.907, intra-class correlation coefficient (ICC) = 0.970, 95% CI 0.959-0.978). Elimination of any one item in all did not result in a value of Cronbach's α of <0.80. SC-FJS had a high correlation with symptoms (0.67, p < 0.001) and pain (0.60, p < 0.001) domains of KOOS and social functioning (0.66, p < 0.001) domain of SF-36, and it also moderately correlated with function in daily living (0.53, p < 0.001) and function in sport and recreation (0.40, p < 0.001) domains of KOOS, and physical subscale of SF-36 (0.49-0.53, p < 0.001) but had a low (r = 0.20) or not significant (p > 0.05) correlation with mental subscale of SF-36. CONCLUSIONS: SC-FJS demonstrated excellent acceptability, internal consistency, reliability, and construct validity, which can be recommended for patients who underwent joint arthroplasty in Mainland China.

Electrospinning of calcium phosphate-poly (d,l-lactic acid) nanofibers for sustained release of water-soluble drug and fast mineralization.

Calcium phosphate-based biomaterials have been well studied in biomedical fields due to their outstanding chemical and biological properties which are similar to the inorganic constituents in bone tissue. In this study, amorphous calcium phosphate (ACP) nanoparticles were prepared by a precipitation method, and used for preparation of ACP-poly(d,l-lactic acid) (ACP-PLA) nanofibers and water-soluble drug-containing ACP-PLA nanofibers by electrospinning. Promoting the encapsulation efficiency of water-soluble drugs in electrospun hydrophobic polymer nanofibers is a common problem due to the incompatibility between the water-soluble drug molecules and hydrophobic polymers solution. Herein, we used a native biomolecule of lecithin as a biocompatible surfactant to overcome this problem, and successfully prepared water-soluble drug-containing ACP-PLA nanofibers. The lecithin and ACP nanoparticles played important roles in stabilizing water-soluble drug in the electrospinning composite solution. The electrospun drug-containing ACP-PLA nanofibers exhibited fast mineralization in simulated body fluid. The ACP nanoparticles played the key role of seeds in the process of mineralization. Furthermore, the drug-containing ACP-PLA nanofibers exhibited sustained drug release which simultaneously occurred with the in situ mineralization in simulated body fluid. The osteoblast-like (MG63) cells with spreading filopodia were well observed on the as-prepared nanofibrous mats after culturing for 24 hours, indicating a high cytocompatibility. Due to the high biocompatibility, sustained drug release, and fast mineralization, the as-prepared composite nanofibers may have potential applications in water-soluble drug loading and release for tissue engineering.

Amorphous calcium phosphate, hydroxyapatite and poly(d,l-lactic acid) composite nanofibers: Electrospinning preparation, mineralization and in vivo bone defect repair.

Due to the outstanding bioactivity and biocompatibility, calcium phosphate (CaP) based materials have been widely investigated for applications in the biomedical fields. In this study, amorphous calcium phosphate (ACP) nanospheres and hydroxyapatite (HA) nanorods have been prepared and hybridized with poly(d,l-lactic acid) (PLA) to fabricate the composite nanofibers through electrospinning. The as-prepared ACP-PLA and HA-PLA composite nanofibers exhibit favorable mineralization behaviors in simulated body fluid (SBF). In the mineralization process, the ACP nanospheres and HA nanorods play an important role in the formation of HA nanosheets on the surface of composite nanofibers. The ACP-PLA and HA-PLA composite nanofibers show a high biocompatibility. The in vivo bone defect repair properties of the ACP-PLA and HA-PLA composite nanofibers are preliminarily investigated. The as-prepared ACP-PLA and HA-PLA composite nanofibers have promising applications in the biomedical fields.