The Regeneration of Large-Sized and Vascularized Adipose Tissue Using a Tailored Elastic Scaffold and dECM Hydrogels.

A dome-shaped elastic poly(l-lactide-co-caprolactone) (PLCL) scaffold with a channel and pore structure was fabricated by a combinative method of 3D printing technology and the gel pressing method (13 mm in diameter and 6.5 mm in thickness) for patient-specific regeneration. The PLCL scaffold was combined with adipose decellularized extracellular matrix (adECM) and heart decellularized extracellular matrix (hdECM) hydrogels and human adipose-derived stem cells (hADSCs) to promote adipogenesis and angiogenesis. These scaffolds had mechanical properties similar to those of native adipose tissue for improved tissue regeneration. The results of the in vitro real-time PCR showed that the dECM hydrogel mixture induces adipogenesis. In addition, the in vivo study at 12 weeks demonstrated that the tissue-engineered PLCL scaffolds containing the hydrogel mixture (hdECM/adECM (80:20)) and hADSCs promoted angiogenesis and adipose tissue formation, and suppressed apoptosis. Therefore, we expect that our constructs will be clinically applicable as material for the regeneration of patient-specific large-sized adipose tissue.

Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells.

Design of macroporous synthetic grafts that can promote infiltration of cells, their differentiation, and synthesis of bone-specific extracellular matrix is a key determinant for in vivo bone tissue regeneration and repair. In this study, we investigated the effect of the microarchitecture of the scaffold on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Poly(ethylene glycol) diacrylate-co-N-acryloyl 6-aminocaproic acid cryogels were fabricated to have either a pore network consisting of cellular, randomly oriented pores (termed 'spongy') or a pore network consisting of lamellar columns (termed 'columnar'), with both cryogel types showing a similar porosity. Both spongy and columnar cryogels supported comparable levels of cell viability and proliferation of hMSCs in vitro. However, spongy cryogels promoted osteogenic differentiation to a greater extent than their columnar counterparts, as evidenced by increased alkaline phosphatase activity and osteoblastic gene expression over 21 days post culture. Leveraging upon our previous work, we further evaluated the ability of these synthetic scaffolds in conjunction with mineralisation to promote ectopic bone formation upon subcutaneous implantation in nude rats. Mineralised spongy and columnar cryogels, both in the presence and absence of exogenous hMSCs, promoted ectopic bone formation in vivo. No such bone formation was observed in acellular cryogels devoid of mineralisation, with extensive host cell infiltration and vascularisation in columnar cryogels, and negligible infiltration into spongy cryogels. Our results thus present a novel method to tune the microarchitecture of porous polymeric scaffolds, in addition to suggesting their efficacy as synthetic bone grafts.

Novel Chitosan Dermal Filler with Enhanced Moldability and Elasticity.

Currently, dermal fillers are largely based on commercialized cross-linked hyaluronic acid (HA) injections, which require a large injection force. Additionally, HA can be easily decomposed by enzymes, and HA-treated tissues present a risk of developing granuloma. In this study, a chitosan-based dermal filler is presented that operates on a liquid-to-gel transition and allows the injection force to be kept ≈4.7 times lower than that required for HA injections. Evaluation of the physical properties of the chitosan filler indicates high viscoelasticity and recovery rate after gelation at 37 °C. Furthermore, in an in vivo evaluation, the liquid injection-type chitosan filler transitions to a gel state within 5 min after injection into the body, and exhibits a compressive strength that is ≈2.4 times higher than that of cross-linked HA. The filler also exhibits higher moldability and maintains a constant volume in the skin for a longer time than the commercial HA filler. Therefore, it is expected that the chitosan filler will be clinically applicable as a novel material for dermal tissue restoration and supplementation.

Skin Regeneration with a Scaffold of Predefined Shape and Bioactive Peptide Hydrogels.

We developed a highly elastic customized scaffold for soft tissue regeneration and combined them with bioactive hydrogels with stem cell-inducing ability. This was done to mimic mechanical properties of native soft tissues and improve the viability of transplanted cells as well as efficiency of tissue regeneration. The proposed study was aimed at evaluating various characteristics of scaffolds and investigating their tissue-regenerating ability. Finger-shaped porous scaffolds were successfully fabricated by an indirect 3D printing of poly (L-lactide-co-ɛ-caprolactone) (PLCL), which provides high elasticity for soft tissue engineering. In addition, a self-assembling peptide hydrogel coupled with substance P (RARADADARARADADA/RARADADARARADADA-substance P, RADA16/RADA16-SP) was used to accelerate angiogenesis and recruit intrinsic mesenchymal stem cells (MSCs). This study included three kinds of groups: Group I = PLCL scaffold with human dermal fibroblasts (HDFs) (P+C), Group II = PLCL scaffold with HDFs and RADA16 (P+C+R), and Group III = PLCL scaffold with HDFs, RADA16, and RADA16-SP (P+C+R+S). The samples were implanted into immunodeficient mice subcutaneously and harvested at 1 and 4 weeks. Tissue regeneration was evaluated by histological analysis with hematoxylin and eosin (H&E) and Masson's trichrome (MT) staining. The images showed that a large number of cells were recruited into the scaffolds, and collagen was deposited in the constructs of the P+C+R+S group. Additionally, recruitment of MSCs, angiogenesis, and collagen were observed by immunofluorescence staining. The results show that the P+C+R+S group had more type I and type III collagen, which are formed in soft tissues, and were deposited on the scaffold compared with the other groups. Moreover, more blood vessels and MSCs were induced in the P+C+R+S group than in those of the P + C and P+C+R groups. Consequently, the results suggest that the construct of the customized porous PLCL scaffold and RADA16/RADA16-SP hydrogel could be a good treatment modality to treat skin defects.

Substance P/dexamethasone-encapsulated PLGA scaffold fabricated using supercritical fluid process for calvarial bone regeneration.

Poly(lactic-co-glycolic acid) (PLGA) scaffolds encapsulated with substance P (SP) and dexamethasone (Dex) by the supercritical CO2 foaming method were fabricated to treat calvarial bone. We compared the release profiles of SP and Dex according to the incorporation methods using encapsulation or dipping. Ninety percent of the SP or Dex molecules in the scaffolds prepared by the encapsulating method were released by day 14 or day 6, respectively. In vivo real-time assays for human mesenchymal stem cell (hMSC) tracking were performed to confirm the MSC recruitment abilities of the scaffolds. The results showed that the optical intensity of the SP-encapsulated group was 2.59 times higher than that of the phosphate-buffered saline group and 1.3 times higher than that of the SP-dipping group. Furthermore, we compared the angiogenesis activity of the scaffolds. In the SP-encapsulated group, 72.9  ± â€…2.6% of the vessels showed matured features by 1 week, and it increased to 82.0  ± â€…4.6% after 4 weeks. We implanted the scaffolds into rat calvarial defects. After 24 weeks, SP- and Dex-encapsulated scaffolds showed 67.1% and 26.2% higher bone formation than those of the Dex-encapsulated group and SP-encapsulated group, respectively, and they formed 36.1% more bone volume compared with the SP- and Dex-dipped scaffolds. Consequently, the results of this study suggest that SP- and Dex-encapsulated scaffolds made by the supercritical CO2 foaming method could be a good treatment modality to treat critical bone defects without cell transplantation by recruiting autologous stem cells and forming new bone tissues. Copyright © 2017 John Wiley & Sons, Ltd.

TGF-ß3 encapsulated PLCL scaffold by a supercritical CO2-HFIP co-solvent system for cartilage tissue engineering.

Mimicking the native tissue microenvironment is critical for effective tissue regeneration. Mechanical cues and sustained biological cues are important factors, particularly in load-bearing tissues such as articular cartilage or bone. Carriers including hydrogels and nanoparticles have been investigated to achieve sustained release of protein drugs. However, it is difficult to apply such carriers alone as scaffolds for cartilage regeneration because of their weak mechanical properties, and they must be combined with other biomaterials that have adequate mechanical strength. In this study, we developed the multifunctional scaffold which has similar mechanical properties to those of native cartilage and encapsulates TGF-ß3 for chondrogenesis. In our previous work, we confirmed that poly(lactide-co-caprolacton) (PLCL) did not foam when exposed to supercritical CO2 below 45°C. Here, we used a supercritical carbon dioxide (scCO2)-1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) co-solvent system to facilitate processing under mild conditions because high temperature causes protein denaturation and decreases bioactivity of the protein. This processing made it possible to fabricate a TGF-ß3 encapsulated elastic porous PLCL scaffold at 37°C. We investigated the tissue regeneration efficiency of the TGF-ß3 encapsulated PLCL scaffold using human adipose-derived stem cells (ADSCs) in vitro and in vivo (Groups; i. PLCL scaffold+Fibrin gel+TGF-ß3, ii. TGF-ß3 encapsulated PLCL scaffold+Fibrin gel, iii. TGF-ß3 encapsulated PLCL scaffold). We evaluated the chondrogenic abilities of the scaffolds at 4, 8, and 12weeks after subcutaneous implantation of the constructs in immune-deficient mice. Based on TGF-ß3 release studies, we confirmed that TGF-ß3 molecules were released by 8weeks and remained in the PLCL matrix. Explants of TGF-ß3 encapsulated scaffolds by a co-solvent system exhibited distinct improvement in the compressive E-modulus and deposition of extracellular matrix. Furthermore, long-term delivery of TGF-ß3 formed a hyaline cartilage-specific lacunae structure and prevented the hypertrophy of differentiated chondrocytes. TGF-ß3 encapsulated PLCL scaffolds would be useful as functional scaffolds for cartilage tissue engineering.

A biocompatible tissue scaffold produced by supercritical fluid processing for cartilage tissue engineering.

Supercritical fluids are used in various industrial fields, such as the food and medical industries, because they have beneficial physical and chemical properties and are also nonflammable and inexpensive. In particular, supercritical carbon dioxide (ScCO(2)) is attractive due to its mild critical temperature, pressure values, and nontoxicity. Poly(L-lactide-co-ɛ-caprolactone) (PLCL), which is a biocompatible, biodegradable, and very elastic polymer, has been used in cartilage tissue engineering. However, organic solvents, such as chloroform or dichloromethane, are usually used for the fabrication of a PLCL scaffold through conventional methods. This leads to a cytotoxic effect and long processing time for removing solvents. To alleviate these problems, supercritical fluid processing is introduced here. In this study, we fabricated a mechano-active PLCL scaffold by supercritical fluid processing for cartilage tissue engineering, and we compared it with a scaffold made by a conventional solvent-casting method in terms of physical and biological performance. Also, to examine the optimum condition for preparing scaffolds with ScCO(2), we investigated the effects of pressure, temperature, and the depressurization rate on PLCL foaming. The PLCL scaffolds produced by supercritical fluid processing had a homogeneously interconnected porous structure, and they exhibited a narrow pore size distribution. Also, there was no cytotoxicity of the scaffolds made with ScCO(2) compared to the scaffolds made by the solvent-pressing method. The scaffolds were seeded with chondrocytes, and they were subcutaneously implanted into nude mice for up to 4 weeks. In vivo accumulation of extracellular matrix of cell-scaffold constructs demonstrated that the PLCL scaffold made with ScCO(2) formed a mature and well-developed cartilaginous tissue compared to the PLCL scaffold formed by solvent pressing. Consequently, these results indicated that the PLCL scaffolds made by supercritical fluid processing offer well-interconnected and nontoxic substrates for cell growth, avoiding problems associated with a solvent residue. This suggests that these elastic PLCL scaffolds formed by supercritical fluid processing could be used for cartilage tissue engineering.