3D-printed porous scaffold promotes osteogenic differentiation of hADMSCs

Objective To explore the role of a three-dimensional (3D)-printed porous titanium alloy scaffold (3D scaffold) in the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) and the underlying mechanism. Methods hADMSCs were divided into control and 3D scaffold groups. The osteogenic differentiation of hADMSCs and expression of osteogenic makers were estimated. Based on the information from published articles, five candidate circular RNAs were selected, and among them, hsa_circ_0019142 showed the most promising results. Finally, control group cells were overexpressed or silenced with the hsa_circ_0019142. Then, Alizarin red S (ARS) staining, calcium content analysis and estimation of alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), and collagen-1 (COL1) were performed to evaluate the role of hsa_circ_0019142 on osteogenic differentiation. Results Osteogenic differentiation of the hADMSCs was significantly higher in the 3D scaffold group than in the control group, as evidenced by ARS staining, increased calcium concentration, and elevated expression of above four osteogenic factors. qPCR revealed that the expression of hsa_circ_0019142 was significantly higher in the 3D scaffold group. Overexpression of hsa_circ_0019142 promoted the osteogenic differentiation of hADMSCs, while knockdown of hsa_circ_0019142 caused the opposite results. Conclusion The 3D-printed scaffold promoted osteogenic differentiation of hADMSCs by upregulating hsa_circ_0019142.

Parathyroid hormone (1-34) promotes the effects of 3D printed scaffold-seeded bone marrow mesenchymal stem cells on meniscus regeneration

Background: Cell-based tissue engineering represents a promising management for meniscus repair and regeneration. The present study aimed to investigate whether the injection of parathyroid hormone (PTH) (1-34) could promote the regeneration and chondroprotection of 3D printed scaffold seeded with bone marrow mesenchymal stem cells (BMSCs) in a canine total meniscal meniscectomy model. Methods: 3D printed poly(e-caprolactone) scaffold seeded with BMSCs was cultured in vitro, and the effects of in vitro culture time on cell growth and matrix synthesis of the BMSCs-scaffold construct were evaluated by microscopic observation and cartilage matrix content detection at 7, 14, 21, and 28 days. After that, the tissue-engineered meniscus based on BMSCs–scaffold cultured for the appropriate culture time was selected for in vivo implantation. Sixteen dogs were randomly divided into four groups: PTH + BMSCs–scaffold, BMSCs–scaffold, total meniscectomy, and sham operation. The regeneration of the implanted tissue and the degeneration of articular cartilage were assessed by gross, histological, and immunohistochemical analysis at 12 weeks postoperatively.Results: In vitro study showed that the glycosaminoglycan (GAG)/DNA ratio and the expression of collagen type II (Col2) were significantly higher on day 21 as compared to the other time points. In vivo study showed that, compared with the BMSCs–scaffold group, the PTH + BMSCs–scaffold group showed better regeneration of the implanted tissue and greater similarity to native meniscus concerning gross appearance, cells composition, and cartilage extracellular matrix deposition. This group also showed less expression of terminal differentiation markers of BMSC chondrogenesis as well as lower cartilage degeneration with less damage on the knee cartilage surface, higher expression of Col2, and lower expression of degeneration markers.Conclusions: Our results demonstrated that PTH (1-34) promotes the regenerative and chondroprotective effects of the BMSCs–3D printed meniscal scaffold in a canine model, and thus their combination could be a promising strategy for meniscus tissue engineering.

Parathyroid hormone (1-34) promotes the effects of 3D printed scaffold-seeded bone marrow mesenchymal stem cells on meniscus regeneration

Background Cell-based tissue engineering represent a promising management for meniscus repair and regeneration. The present study aimed to investigate whether the injection of parathyroid hormone (PTH) (1–34) could promote the regeneration and chondroprotection of 3D printed scaffold seeded with bone marrow mesenchymal stem cells (BMSCs) in a canine total meniscal meniscectomy model. Methods 3D printed poly(e-caprolactone) scaffold seeded with BMSCs was cultured in vitro, and the effects of in vitro culture time on cell growth and matrix synthesis of the BMSCs-scaffold construct were evaluated by microscopic observation and cartilage matrix content detection at 7, 14, 21, and 28 days. After that, the tissue-engineered meniscus based on BMSCs–scaffold cultured for the appropriate culture time was selected for in vivo implantation. Sixteen dogs were randomly divided into four groups: PTH + BMSCs–scaffold, BMSCs–scaffold, total meniscectomy, and sham operation. The regeneration of the implanted tissue and the degeneration of articular cartilage were assessed by gross, histological, and immunohistochemical analysis at 12 weeks postoperatively. Results In vitro study showed that the glycosaminoglycan (GAG)/DNA ratio and the expression of collagen type II (Col2) were significantly higher on day 21 as compared to the other time points. In vivo study showed that, compared with the BMSCs–scaffold group, the PTH + BMSCs–scaffold group showed better regeneration of the implanted tissue and greater similarity to native meniscus with respect to gross appearance, cells composition, and cartilage extracellular matrix deposition. This group also showed less expression of terminal differentiation markers of BMSC chondrogenesis as well as lower cartilage degeneration with less damage on the knee cartilage surface, higher expression of Col2 and lower expression of degeneration markers. Conclusions Our results demonstrated that PTH (1–34) promotes the regenerative and chondroprotective effects of the BMSCs–3D printed meniscal scaffold in a canine model, and thus their combination could be a promising strategy for meniscus tissue engineering.

Synthetic Biphasic Scaffolds versus Microfracture for Articular Cartilage Defects of the Knee: A Retrospective Comparative Study

Objective The purpose of this study was to compare the results of a biphasic synthetic scaffold (TruFit, Smith & Nephew) to microfracture for the treatment of knee cartilage defects and identify patient- and lesion-specific factors that influence outcomes. Design Prospectively collected data from 132 patients (mean age, 41.8 years; 69% male) with isolated chondral or osteochondral femoral defects treated with biphasic synthetic scaffolds ( n = 66) or microfracture ( n = 66) were reviewed. Clinical outcomes were evaluated longitudinally over 5 years with the Short Form–36 (SF-36), Activities of Daily Living of the Knee Outcome Survey (KOS-ADL), International Knee Documentation Committee (IKDC), and Marx Activity Scale. Cartilage-sensitive magnetic resonance imaging (MRI) was performed to evaluate osseous integration and cartilage fill in a subgroup of patients. Multivariate regression analysis was used to identify predictors of clinical outcomes within the scaffold group. Results Both groups demonstrated clinically significant improvements in knee clinical scores over 5 years ( P < 0.01). There were no significant differences in KOS-ADL and IKDC scores between groups up to 5 years postoperatively. Marx activity level scores in the microfracture group declined over time, while significant improvements in activity level scores were observed in the scaffold group over 5 years ( P < 0.01). Good-quality tissue fill and cartilage isointensity were more often observed in the scaffold group compared with the microfracture group, particularly with longer time intervals. Increasing age, high body mass index, prior microfracture, and traumatic etiology were predictors for inferior outcomes in the scaffold group. Conclusions Activity level and MRI appearance following treatment of cartilage lesions with the biphasic synthetic scaffold were superior to microfracture over time in this nonrandomized, retrospective comparison.

Autologous stem cell-derived chondrocyte implantation with bio-targeted microspheres for the treatment of osteochondral defects

Background Chondral injury is a common problem around the world. Currently, there are several treatment strategies for these types of injuries. The possible complications and problems associated with conventional techniques lead us to investigate a minimally invasive and biotechnological alternative treatment. Combining tissue-engineering and microencapsulation technologies provide new direction for the development of biotechnological solutions. The aim of this study is to develop a minimal invasive tissue-engineering approach, using bio-targeted microspheres including autologous cells, for the treatment of the cartilage lesions. Method In this study, a total of 28 sheeps of Akkaraman breed were randomly assigned to one of the following groups: control (group 1), microfracture (group 2), scaffold (group 3), and microsphere (group 4). Microspheres and scaffold group animals underwent adipose tissue collection prior to the treatment surgery. Mesenchymal cells collected from adipose tissue were differentiated into chondrocytes and encapsulated with scaffolds and microspheres. Osteochondral damage was conducted in the right knee joint of the sheep to create an animal model and all animals treated according to study groups. Results Both macroscopic and radiologic examination showed that groups 3 and 4 have resulted better compared to the control and microfracture groups. Moreover, histologic assessments indicate hyaline-like cartilage formations in groups 3 and 4. Conclusion In conclusion, we believe that the bio-targeted microspheres can be a more effective, easier, and safer approach for cartilage tissue engineering compared to previous alternatives.

Effect of Collagen Scaffold With Bcl-2-Modified Adipose-Derived Stem Cells on Diabetic Mice Wound Healing

This study aimed at evaluating the effects of collagen scaffold with Bcl-2-modified adipose-derived stem cells (ADSCs) on wound repair in streptozotocin-induced diabetic mice. A round full thickness skin defect with a diameter of 7 mm was made in the mice model. The experimental mice were divided into 4 groups (n = 12 each): group A (control group), group B (scaffold group), group C (ADSCs-scaffold group), and group D (Bcl-2-ADSCs-scaffold group). On days 3, 7, 10, and 14 after surgery, characteristics of wound healing was observed, and wound tissues were sampled for histology characteristics via hematoxylin-eosin staining and immunohistochemical staining. Compared with other groups, the wound healing rate was significantly higher in group D a week after operation ( P < .05). On the seventh day postoperation, group D exhibited higher blood vessel in the wounds granulation tissue than other groups according to results of hematoxylin-eosin staining and immunohistochemistry. In conclusion, these findings demonstrated that collagen scaffold with Bcl-2 modified ADSCs may effectively improve the wound healing process in diabetic mice.

Aptamer-Functionalized Bioscaffold Enhances Cartilage Repair by Improving Stem Cell Recruitment in Osteochondral Defects of Rabbit Knees

Background: Recruitment of endogenous stem cells has been considered an alternative to cell injection/implantation in articular cartilage repair. Purpose: (1) To develop a cartilage tissue-engineering scaffold with clinically available biomaterials and functionalize the scaffold with an aptamer (Apt19s) that specifically recognizes pluripotent stem cells. (2) To determine whether this scaffold could recruit joint-resident mesenchymal stem cells (MSCs) when implanted into an osteochondral defect in a rabbit model and to examine the effects of cartilage regeneration. Study Design: Controlled laboratory study. Methods: The reinforced scaffold was fabricated by embedding a silk fibroin sponge into silk fibroin/hyaluronic acid–tyramine hydrogel and characterized in vitro. A cylindrical osteochondral defect (3.2 mm wide × 4 mm deep) was created in the trochlear grooves of rabbit knees. The rabbits were randomly assigned into 3 groups: Apt19s-functionalized scaffold group, scaffold-only group, and control group. Animals were sacrificed at 6 and 12 weeks after transplantation. Repaired tissues were evaluated via gross examination, histologic examination, and immunohistochemistry. Results: In vitro, this aptamer-functionalized scaffold could recruit bone marrow–derived MSCs and support cell adhesion. In vivo, the aptamer-functionalized scaffold enhanced cell homing in comparison with the aptamer-free scaffold. The aptamer-functionalized scaffold group also exhibited superior cartilage restoration when compared with the scaffold-only group and the control group. Conclusion: The Apt19s-functionalized scaffold exhibited the ability to recruit MSCs both in vitro and in vivo and achieved a better outcome of cartilage repair than the scaffold only or control in an osteochondral defect model. Clinical Relevance: The findings demonstrate a promising strategy of using aptamer-functionalized bioscaffolds for restoration of chondral/osteochondral defects via aptamer-introduced homing of MSCs.

Bone regeneration in Ds-Red pig calvarial defect using allogenic transplantation of EGFP-pMSCs – a comparison of host cells and seeding cells in the scaffold

BackgroundCells, scaffolds, and factors are the triad of regenerative engineering; however, it is difficult to distinguish whether cells in the regenerative construct are from the seeded cells or host cells via the host blood supply. We performed a novel in vivo study to transplant enhanced green fluorescent pig mesenchymal stem cells (EGFP-pMSCs) into calvarial defect of DsRed pigs. The cell distribution and proportion were distinguished by the different fluorescent colors through the whole regenerative period.Method/ResultsEight adult domestic Ds-Red pigs were treated with five modalities: empty defects without scaffold (group 1); defects filled only with scaffold (group 2); defects filled with osteoinduction medium-loaded scaffold (group 3); defects filled with 5 × 103 cells/scaffold (group 4); and defects filled with 5 × 104 cells/scaffold (group 5). The in vitro cell distribution, morphology, osteogenic differentiation, and fluorescence images of groups 4 and 5 were analyzed. Two animals were sacrificed at 1, 2, 3, and 4 weeks after transplantation. The in vivo fluorescence imaging and quantification data showed that EGFP-pMSCs were represented in the scaffolds in groups 4 and 5 throughout the whole regenerative period. A higher seeded cell density resulted in more sustained seeded cells in bone regeneration compared to a lower seeded cell density. Host cells were recruited by seeded cells if enough space was available in the scaffold. Host cells in groups 1 to 3 did not change from the 1st week to 4th week, which indicates that the scaffold without seeded cells cannot recruit host cells even when enough space is available for cell ingrowth. The histological and immunohistochemical data showed that more cells were involved in osteogenesis in scaffolds with seeded cells.ConclusionOur in vivo results showed that more seeded cells recruit more host cells and that both cell types participate in osteogenesis. These results suggest that scaffolds without seeded cells may not be effective in bone transplantation.

Adipose-Derived Stem Cells Ameliorate Ischemia-Reperfusion Injury in a Rat Skin Free Flap Model

Background Ischemia-reperfusion (I/R) injury is inevitable during free tissue transfers. When the period of ischemia exceeds the tissue tolerance, it causes necrosis and flap failure. The aim of this study was to investigate the effects of adipose-derived stem cells (ASCs) embedded in a collagen type I scaffold on the survival of free skin flaps to counteract I/R injury. Methods Left superficial caudal epigastric skin flaps (3 × 6 cm) were performed in 28 Wistar rats that were divided into four groups. The flaps elevated in the animals of the control group did not suffer any ischemic insult, and the vascular pedicle was not cut. All other flaps were subjected to 8 hours of ischemia prior to revascularization: I/R control group (8 hours of ischemia), I/R scaffold group (8 hours of ischemia + collagen type I scaffold), and I/R scaffold–ASCs group (8 hours of ischemia + collagen type I scaffold with rat ASCs embedded). Transit-time ultrasound blood flow measurements were performed. After 7 days, the areas of flap survival were measured and tissues were stained with hematoxylin/eosin and Masson's trichrome stain for histological analysis. Results The mean percentage flap survival area was significantly higher in the ASCs-treated flaps (I/R scaffold–ASCs group) compared with the ischemic controls (I/R control group and I/R scaffold group). Higher vascular proliferation and lower severity of necrosis and inflammatory changes were seen histologically in the samples of the ASCs-treated group. No significant difference in blood flow was detected between groups. Conclusion Subcutaneous administration of ASCs embedded on a collagen type I scaffold reduces tissue damage after I/R injury in microvascular free flaps.

Abstract 320: Delivery of Hepatocyte Growth Factor mRNA From Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease

Biological approaches to augment angiogenesis are promising for treatment of peripheral arterial disease (PAD). We propose the use of scaffold-based modified mRNA (mmRNA) delivery as a favorable approach for transient, localized gene delivery. We hypothesized that hepatocyte growth factor (HGF) mmRNA-seeded nanofibrillar scaffolds will enable localized and temporally controlled delivery of mmRNA, leading to augmentation of angiogenesis in a murine model of PAD. To establish the efficacy of mmRNA therapy, mmRNA encoding green fluorescence protein (GFP) was used as a fluorescent reporter for quantification of transfection efficiency. Aligned nanofibrillar collagen scaffolds were loaded with mmRNA and lipofectamine transfection agent. The temporal kinetics of mmRNA release into media was measured by ribogreen assay. To determine the transfection efficiency, human fibroblasts were cultured on the aligned nanofibrillar scaffolds, or on tissue culture plastic, and the efficiency of transfection was measured for up to 7 days and assayed for GFP expression. Based on ribogreen assay, the cumulative release of GFP mmRNA over the course of 14 days was 235 ng/cm scaffold. In vitro transfection efficiency on aligned scaffolds (75%) was markedly higher than on tissue culture plastic (45%) after 24h. The persistence of cellular transfection as quantified by western blotting showed GFP expression >5 days post-transfection. Next, to demonstrate therapeutic efficacy for treatment of PAD, scaffolds releasing HGF or GFP mmRNA were transplanted to the site of the murine ischemic hindlimb. At the end of the 14 day experiment, laser Doppler spectroscopy showed that HGF mmRNA scaffold group had a higher mean perfusion ratio (0.32 ±0.10) than the GFP mmRNA scaffold group (0.23±0.14), suggesting that HGF-scaffolds improved blood perfusion. In summary, these data suggest that HGF mmRNA-releasing scaffolds marked improved blood perfusion in a murine model of PAD.