Evaluation of osteoconductive effect of polycaprolactone (PCL) scaffold treated with fibronectin on adipose-derived mesenchymal stem cells (AD-MSCs).

What's in a Name?

Three-dimensional (3D)-printed breast scaffolds have attracted increased attention for soft tissue reconstruction. However, the polymeric porous scaffolds commonly cause fibrous tissue ingrowth due to their limited immunomodulatory capabilities. In this study, we integrated polycaprolactone (PCL) scaffolds with adipose-derived mesenchymal stem cell (ADSC) exosome-laden Gelatin Methacrylate (GelMA) hydrogels (Exos@GelMA+PCL) to promote macrophage M2 polarization and adipose regeneration. The biohybrid scaffolds exhibited sustained Exo release, with a cumulative release of >80% by day 14. Internalized Exos enhanced RAW264.7 macrophage M2 polarizationin vitro, as confirmed by immunofluorescence and real-time quantitative PCR. Conditioned medium from scaffold-macrophage cocultures enhanced the proliferation, migration, and adipogenic differentiation of ADSCs.In vivo, Exos@GelMA+PCL biohybrid scaffolds significantly increased the proportion of M2 macrophages compared to controls (GelMA+PCL and PCL scaffolds). At 12 weeks, the biohybrid scaffolds achieved markedly higher adipose tissue area percentages (46.26 ± 4.55%) compared to GelMA+PCL scaffolds (23.76 ± 1.90%) and PCL scaffolds (26.14 ± 2.55%). This strategy offers an innovative immunomodulatory approach to enhance soft tissue regeneration in breast reconstruction by regulating the microenvironment.

Biologically modified electrospun polycaprolactone nanofibrous scaffold promotes osteogenic differentiation

Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells into Polycaprolactone (PCL) Scaffold

Bone tissue as a dynamic tissue is able to repair its minor injuries, however, sometimes the repair cannot be completed by itself due to the size of lesion. In such cases, the best treatment could be bone tissue engineering. The use of stem cells in skeletal disorders to repair bone defects has created bright prospects. On the other hand, changes in the expression level of microRNAs (miRs) can lead to the commitment of mesenchymal stem cells (MSCs) to cell lineage. Many studies reported that post-transcriptional regulations by miRNAs are involved in all stages of osteoblast differentiation. After the preparing adipose tissue-derived mesenchymal stem cells, the target cells from the third passage were cultured in two groups, transfected MSCs with miR-27a-3p (DM.C + P) and control group. In different times, 7 and 14days after culture, differentiation of these cells into osteoblast were measured using various techniques including the ALP test and calcium content test, Alizarin Red staining, Immunocytochemistry technique (ICC). Also, the relative expression of bone differentiation marker genes including Osteonectin (ON), Osteocalcin (OC), RUNX Family Transcription Factor 2 (RUNX2), Collagen type I alpha 1 (COL1) was investigated by real-time RT PCR. In comparison with control groups, overexpression of miR-27a-3p in transfected cells resulted in a significant increase in the expression of bone markers genes (ON, OC, RUNX2, COL1), alkaline phosphatase (ALP) activity, and calcium content (p < 0.05). In addition, the results obtained from ICC technique showed that osteocalcin protein is expressed at the surface of bone cells. Furthermore, the expression of APC, as a target of miR-27a-3p, decreased in transfected cells. Our data suggest that miR-27a-3p may positively regulates adipose tissue-derived mesenchymal stem cell differentiation into bone by targeting APC and activating the Wnt/b-catenin pathway.

The damaged articular cartilage has limited self-regeneration capacity because of the absence of blood vessels, lymphatics, and nerves. Cartilage transplantation is, hence, a popular method used to treat this disease. However, sources of autograft and allogenic cartilage for transplantation are limited. Therefore, this study aims to suggest a simple method to produce engineered cartilage from human adipose-derived mesenchymal stem cells (ADSCs) and poly (ε-caprolactone) (PCL) scaffolds. ADSCs were isolated and expanded from fat tissues according to published protocols. PCL-porous scaffolds were produced from PCL with 5 × 5 × 0.6mm3 with 200-400μ m pore sizes. ADSCs were seeded on the PCL scaffolds at three different densities (104, 105, 106 cells per scaffold). The adherence of ADSCs on the surface of PCL scaffolds was evaluated based on an immunostaining assay to determine the presence of ADSCs. The cell proliferation on PCL scaffolds was determined by MTT assay. The complexity in ADSCs and PCL scaffolds was induced to cartilage using a chondrogenesis medium. The engineered cartilage was characterized by the accumulation of proteoglycan and aggrecan by Safranin O staining assay. Their structures were evaluated using an H-E staining assay. Finally, these engineered cartilage tissues were transplanted into mice to assess cartilage maturation when compared to natural cartilage. The results showed that the engineered cartilage tissues could be successfully produced by cultures of ADSCs on poly ε-caprolactone scaffolds in combination with chondrogenesis medium. The suitable density of ADSCs was 106 cells/per scaffold of 5 × 6 × 0.6mm3 with pore size from 200 to 400μ m. The results showed that an in vitro cartilage tissue was created from ADSCs and PCL scaffold. The cartilage tissue exists in the mice for 6months.

Cumulative Evidence That Mesenchymal Stem Cells Promote Healing of Perianal Fistulas of Patients With Crohn's Disease–Going From Bench to Bedside

Polycaprolactone (PCL) is a bioresorbable polymer with potential applications for bone and cartilage repair. In this work, three-dimensional (3D) and porous PCL scaffolds were designed and fabricated via selective laser sintering (SLS). The aim of this study was to evaluate the osteogenic potential of porcine adipose-derived stem cells (pASCs) in a laser-sintered PCL (lsPCL) scaffold. The character of the lsPCL scaffold was evaluated. The pore size and the microstructure were observed by SEM. The pASCs were harvested and isolated from pig inguinal area. Then, the lsPCL scaffold was seeded with ASCs and cultured in osteogenic medium for 0 and 14 days. Cell proliferation was measured by MTS. Alkaline phosphatase activity (ALP) was detected using biochemical methods. SEM was used to observe the interaction between scaffold and cell. An energy dispersive spectrum (EDS) was used to analyze the mineralization in each group. Porosity was around 83%; pore size was around 300–400 µm. Both MTS and ALP showed significant increase after subcultivation in osteogenic medium for 14 days. SEM detailed that the pASCs cell can attach well to the lsPCL scaffold. The energy dispersive spectrum (EDS) also demonstrated calcium deposits around pASCs after osteo-induction for 14 days. In contrast, no mineralization was found around ASCs after osteo-induction of 0 days. In conclusion, the laser-sintered PCL is a suitable scaffold for the proliferation of ASCs. The ASCs were also well differentiated into osteoblasts in the 3D, porous, laser-sintered PCL scaffold.

Loss of motor and sensory function as a result of neuronal cell death and axonal degeneration are the hallmarks of spinal cord injury. To overcome the hurdles and achieve improved functional recovery multiple aspects, it must be taken into account. Tissue engineering approaches by coalescing biomaterials and stem cells offer a promising future for treating spinal cord injury. Here we investigated human endometrial stem cells (hEnSCs) as our cell source. Electrospun poly ε-caprolactone (PCL) scaffolds were used for hEnSC adhesion and growth. Scanning electron microscopy (SEM) confirmed the attachment and survival of stem cells on the PCL scaffolds. The scaffold-stem cell construct was transplanted into the hemisected spinal cords of adult male rats. Crocin, an ethanol-extractable component of Crocus sativus L., was administered to rats for 15 consecutive days post injury. Neurite outgrowth and axonal regeneration were investigated using immunohistochemical staining for neurofilament marker NF-H and luxol-fast blue (LFB) staining, respectively. TNF-α staining was performed to determine the inflammatory response in each group. Functional recovery was assessed via the Basso-Beattie-Bresnahan (BBB) scale. Results showed that PCL scaffolds seeded with hEnSCs restored the continuity of the damaged spinal cord and decreased cavity formation. Additionally, hEnSC-seeded scaffolds contributed to the functional recovery of the spinal cord. Hence, hEnSC-seeded PCL scaffolds may serve as promising transplants for spinal cord tissue engineering purposes. Furthermore, crocin had an augmenting effect on spinal cord regeneration and proved to exert neuroprotective effects on damaged neurons and may be further studied as a promising drug for spinal cord injury.

Multipotent mesenchymal stem cells (MSCs) and MSC sheets have effective potentials of bone regeneration. Composite polymer/ceramic scaffolds such as poly-ε-caprolactone (PCL)/β-tricalcium phosphate (β-TCP) are widely used to repair large bone defects. The present study investigated the in vitro osteogenic potential of canine adipose-derived MSCs (Ad-MSCs) and Ad-MSC sheets. Composite PCL/β-TCP scaffolds seeded with Ad-MSCs or wrapped with osteogenic Ad-MSC sheets (OCS) were also fabricated and their osteogenic potential was assessed following transplantation into critical-sized bone defects in dogs. The alkaline phosphatase (ALP) activity of osteogenic Ad-MSCs (O-MSCs) and OCS was significantly higher than that of undifferentiated Ad-MSCs (U-MSCs). The ALP, runt-related transcription factor 2, osteopontin, and bone morphogenetic protein 7 mRNA levels were upregulated in O-MSCs and OCS as compared to U-MSCs. In a segmental bone defect, the amount of newly formed bone was greater in PCL/β-TCP/OCS and PCL/β-TCP/O-MSCs/OCS than in the other groups. The OCS exhibit strong osteogenic capacity, and OCS combined with a PCL/β-TCP composite scaffold stimulated new bone formation in a critical-sized bone defect. These results suggest that the PCL/β-TCP/OCS composite has potential clinical applications in bone regeneration and can be used as an alternative treatment modality in bone tissue engineering.

Heterogeneity of mesenchymal stem cells (MSCs) influences the cell therapy outcome and the application in tissue engineering. Also, the application of subpopulations of MSCs in cartilage regeneration remains poorly characterized. CD146+ MSCs are identified as the natural ancestors of MSCs and the expression of CD146 are indicative of greater pluripotency and self-renewal potential. Here, we sorted a CD146+ subpopulation from adipose-derived mesenchymal stem cells (ADSCs) for cartilage regeneration.Methods: CD146+ ADSCs were sorted using magnetic activated cell sorting (MACS). Cell surface markers, viability, apoptosis and proliferation were evaluated in vitro. The molecular signatures were analyzed by mRNA and protein expression profiling. By intra-articular injections of cells in a rat osteochondral defect model, we assessed the role of the specific subpopulation in cartilage microenvironment. Finally, CD146+ ADSCs were combined with articular cartilage extracellular matrix (ACECM) scaffold for long term (3, 6 months) cartilage repair.Results: The enriched CD146+ ADSCs showed a high expression of stem cell and pericyte markers, good viability, and immune characteristics to avoid allogeneic rejection. Gene and protein expression profiles revealed that the CD146+ ADSCs had different cellular functions especially in regulation inflammation. In a rat model, CD146+ ADSCs showed a better inflammation-modulating property in the early stage of intra-articular injections. Importantly, CD146+ ADSCs exhibited good biocompatibility with the ACECM scaffold and the CD146+ cell-scaffold composites produced less subcutaneous inflammation. The combination of CD146+ ADSCs with ACECM scaffold can promote better cartilage regeneration in the long term.Conclusion: Our data elucidated the function of the CD146+ ADSC subpopulation, established their role in promoting cartilage repair, and highlighted the significance of cell subpopulations as a novel therapeutic for cartilage regeneration.

Bone tissue engineering (BTE) is now a promising research issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self renewal and multi-lineage differentiation. Unlike embryonic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells (BMSCs) are the earliest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical application. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stem cells (ASCs), is found to be more suitable in clinical application because of high stem cells yield from lipoaspirates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated because most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation potential. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.

Bone tissue engineering is an emerging field providing viable substitutes for bone regeneration. Recent advances have allowed scientists and engineers to develop scaffolds for guided bone growth. However, success requires scaffolds to have specific macroscopic geometries and internal architectures conducive to biological and biophysical functions. Freeform fabrication provides an effective process tool to manufacture three-dimensional porous scaffolds with complex shapes and designed properties. A novel precision extruding deposition (PED) technique was developed to fabricate polycaprolactone (PCL) scaffolds. It was possible to manufacture scaffolds with a controlled pore size of 350 µm with designed structural orientations using this method. The scaffold morphology, internal micro-architecture and mechanical properties were evaluated using scanning electron microscopy (SEM), micro-computed tomography (micro-CT) and mechanical testing, respectively. An in vitro cell–scaffold interaction study was carried out using primary fetal bovine osteoblasts. Specifically, the cell proliferation and differentiation was evaluated by Alamar Blue assay for cell metabolic activity, alkaline phosphatase activity and osteoblast production of calcium. An in vivo study was performed on nude mice to determine the capability of osteoblast-seeded PCL to induce osteogenesis. Each scaffold was implanted subcutaneously in nude mice and, following sacrifice, was explanted at one of a series of time intervals. The explants were then evaluated histologically for possible areas of osseointegration. Microscopy and radiological examination showed multiple areas of osseous ingrowth suggesting that the osteoblast-seeded PCL scaffolds evoke osteogenesis in vivo. These studies demonstrated the viability of the PED process to fabricate PCL scaffolds having the necessary mechanical properties, structural integrity, and controlled pore size and interconnectivity desired for bone tissue engineering.

Tissue-Engineered Bone With 3-Dimensionally Printed β-Tricalcium Phosphate and Polycaprolactone Scaffolds and Early Implantation: An In Vivo Pilot Study in a Porcine Mandible Model

We have previously demonstrated that adipose-derived stem cells (ASC) enhance healing of craniofacial bone in vivo. The use of scaffolds in large bone defects can further enhance the healing capacity of ASC. In addition, the presence of fully differentiated cells might affect ASC migration/proliferation into scaffolds. In this study, we have evaluated the in vitro capacity of ASC isolated from fat of GFP transgenic pigs to migrate into polycaprolactone (PCL) scaffolds conjugated with FGF or BMP2 with or without combinations of ASC with pig endothelial and/or neuronal cells. The ASC were extracted from the back fat of a GFP boar while the endothelial cells (ENDO) were extracted from the aorta a newborn piglet and the neurons (NEU) from the brain of a 37 days fetus. All cells were used at passage 3. In a 24-well plate, approximately 10 000 cells (only ASC, ENDO, or NEU, or equal combination of ASC+ENDO, ASC+NEU, or ASC+ENDO+NEU) were plated. The cells were grown for 3 days before adding the scaffolds in the wells. The scaffolds were treated with basic human FGF (25 ng mg–1 of scaffold), porcine BMP2 (400 ng mg–1 of scaffold), or no treatment (CTR). The scaffolds were then added to the plate and the medium was changed every 3 days. Using an Olympus IX71 microscope with automatic stage several pictures at 100× and 200× magnifications were taken and time-lapse visualisation of the movement of cells into the scaffold was performed during the first week. At 24 days the scaffolds were fixed with formalin. In order to estimate cell size and visualise non-GFP cells all scaffolds were treated with DAPI. Final photographs of the whole scaffold using a multiple alignment images features at 40× magnification were performed using GFP and DAPI filter plus brightfield. The figures were analysed with ImageJ to quantify the area of GFP and DAPI in the scaffolds. Percentage GFP and DAPI over scaffold area were calculated. Data were analysed using a Proc ANOVA of SAS, with cells and treatment as fixed effects and well as random. At 1 week from the beginning of the trial, we observed ASC cells on the scaffolds. The time-lapse showed ASC actively moving on the scaffold surface and entering into its micropores. The quantification of GFP and DAPI showed a significant larger percentage area covered by GFP cells in scaffolds treated with FGF (P &lt; 0.05) compared to CTR or BMP2 (32 ± 4.6 vs 12.8 ± 9.1 and 3.8 ± 0.1). We observed a numerical lower area covered by GFP in scaffolds treated with BMP2 compared to CTR. The same trends were observed for the area covered by DAPI with a numerically lower colonization of the scaffolds by ENDO when treated with BMP2 vs CTR. Data showed that ASC, as well ENDO and NEU, are able to home into PCL scaffolds. The conjugation of the scaffolds with FGF has a positive effect on ASC migration, with no effects of BMP2. Data support the use of PCL scaffolds to enhance the bone healing capacity in vivo of ASC with a likely further improvement if scaffolds are treated with FGF before implantation.