Calcium deposition in photocrosslinked poly(Pro‐Hyp‐Gly) hydrogels encapsulated rat bone marrow stromal cells

Reproducing the features of the extracellular matrix is important for fabricating three‐dimensional (3D) scaffolds for tissue regeneration. A collagen‐like polypeptide, poly(Pro‐Hyp‐Gly), is a promising material for 3D scaffolds because of its excellent physical properties, biocompatibility, and biodegradability. In this paper, we present a novel photocrosslinked poly(Pro‐Hyp‐Gly) hydrogel as a 3D scaffold for simultaneous rat bone marrow stromal cell (rBMSC) encapsulation. The hydrogels were fabricated using visible‐light photocrosslinking at various concentrations of methacrylated poly(Pro‐Hyp‐Gly) (20–50 mg/ml) and irradiation times (3 or 5 min). The results show that the rBMSCs encapsulated in the hydrogels survived 7 days of incubation. Calcium deposition on the encapsulated rBMSCs was assessed with scanning electron microscope observation, Alizarin Red S, and von Kossa staining. The most strongly stained area was observed in the hydrogel formed with 30 mg/ml of methacrylated poly(Pro‐Hyp‐Gly) with 5‐min irradiation. These findings demonstrate that poly(Pro‐Hyp‐Gly) hydrogels support rBMSC viability and differentiation, as well as demonstrating the feasibility of using poly(Pro‐Hyp‐Gly) hydrogels as a cytocompatible, biodegradable 3D scaffold for tissue regeneration.     

SIKVAV‐modified highly superporous PHEMA scaffolds with oriented pores for spinal cord injury repair

The architecture and mechanical properties of a scaffold for spinal cord injury treatment must provide tissue integration as well as effective axonal regeneration. Previous work has demonstrated the cell‐adhesive and growth‐promoting properties of the SIKVAV (Ser–Ile–Lys–Val–Ala–Val)‐modified highly superporous poly(2‐hydroxethyl methacrylate) (PHEMA) hydrogels. The aim of the current study was to optimize the porosity and mechanical properties of this type of hydrogel in order to develop a suitable scaffold for the repair of spinal cord tissue. Three types of highly superporous PHEMA hydrogels with oriented pores of ~60 µm diameter, porosities of 57–68% and equivalent stiffness characterized by elasticity moduli in the range 3–45 kPa were implanted into a spinal cord hemisection, and their integration into the host tissue, as well as the extent of axonal ingrowth into the scaffold pores, were histologically evaluated. The best tissue response was found with a SIKVAV‐modified PHEMA hydrogel with 68% porosity and a moderate modulus of elasticity (27 kPa in the direction along the pores and 3.6 kPa in the perpendicular direction). When implanted into a spinal cord transection, the hydrogel promoted tissue bridging as well as aligned axonal ingrowth. In conclusion, a prospective oriented scaffold architecture of SIKVAV‐modified PHEMA hydrogels has been developed for spinal cord injury repair; however, to develop an effective treatment for spinal cord injury, multiple therapeutic approaches are needed. Copyright © 2013 John Wiley & Sons, Ltd.     

Culture of preantral follicles in poly(ethylene) glycol‐based,three‐dimensional hydrogel: a relationship between swelling ratio and follicular developments

This study was undertaken to examine how the softness of poly(ethylene) glycol (PEG)‐based hydrogels, creating a three‐dimensional (3D) microenvironment, influences the in vitro growth of mouse ovarian follicles. Early secondary, preantral follicles of 2 week‐old mice were cultured in a crosslinked four‐arm PEG hydrogel. The hydrogel swelling ratio, which relates to softness, was modified within the range 25.7–15.5 by increasing the reactive PEG concentration in the precursor solution from 5% to 15% w/v, but it did not influence follicular growth to form the pseudoantrum (60–80%; p = 0.76). Significant (p < 0.04) model effects, however, were detected in the maturation and developmental competence of the follicle‐derived oocytes. A swelling ratio of > 21.4 yielded better oocyte maturation than other levels, while the highest competence to develop pronuclear and blastocyst formation was detected at 20.6. In conclusion, gel softness, as reflected in swelling ratio, was one of the essential factors for supporting folliculogenesis in vivo within a hydrogel‐based, 3D microenvironment. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

Collagen–chitosan–laminin hydrogels for the delivery of insulin‐producing tissue

Islet transplantation is an emerging strategy for treating patients with type 1 diabetes mellitus. Although the proof of concept for cellular replacement therapy in diabetes has been firmly established, vascularity of the transplant site and the long‐term survival and function of transplanted islets remains suboptimal. In the present study, human circulating angiogenic cells (CACs) and porcine islet cells embedded in collagen–chitosan hydrogels, with and without laminin, were investigated as potential engineered biomaterials for the treatment of type 1 diabetes. Hydrogels were evaluated in vitro for their physical properties (compression, degradation, porosity and wettability) and cell compatibility. Increasing the chitosan content in the collagen‐based hydrogel resulted in increased stiffness (p ≤ 0.04) and time to gelation (p < 0.001), but reduced porosity (from 22–28% to 16–19%). The material design formulations (10:1 vs 20:1 collagen:chitosan ratio) directly affected the cell properties. The viability of both human CACs and porcine islets embedded in the 20:1 collagen–chitosan matrix was higher at 24 h compared to the 10:1 formulation. For islet function, glucose stimulation indices for the 20:1 formulation at 24 h compared favourably with values reported in the literature, more so than the 10:1 formulations. While laminin improved the short‐term viability of CACs, its presence did not confer any benefit to islet viability or function. Overall, the design features outlined in this study provided the degree of control required to establish viable tissue with potential for islet transplantation and neovascularization. Copyright © 2013 John Wiley & Sons, Ltd.     

Preparation of three‐dimensional macroporous chitosan–gelatin B microspheres and HepG2‐cell culture

Chitosan–gelatin B microspheres with an open, interconnected, highly macroporous (100–200 µm) structure were prepared via a three‐step protocol combining freeze‐drying with an electrostatic and ionic cross‐linking method. Saturated tripolyphosphate ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure and to improve the biostability of the chitosan–gelatin B microspheres, with N‐(3‐dimethylaminopropyl)‐N′‐ethyl‐carbodiimide/N‐hydroxysuccinimide as a second crosslinking agent to react with gelatin A and fixed chitosan–gelatin B microspheres to attain improved biocompatibility. Water absorption of the three‐dimensional macroporous chitosan–gelatin B microspheres (3D‐P‐CGMs) was 12.84, with a porosity of 85.45%. In vitro lysozyme degradation after 1, 3, 5, 7, 10, 14, and 21 days showed improved biodegradation in the 3D‐P‐CGMs. The morphology of human hepatoma cell lines (HepG2 cells) cultured on the 3D‐P‐CGMs was spherical, unlike that of cells cultured under traditional two‐dimensional conditions. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3D‐P‐CGMs. HepG2 cells were able to migrate inside through the pore. Cell proliferation and levels of albumin and lactate dehydrogenase suggested that the 3D‐P‐CGMs could provide a larger specific surface area and an appropriate microenvironment for cell growth and survival. Hence, the 3D‐P‐CGMs are eminently suitable as macroporous scaffolds for cell cultures in tissue engineering and cell carrier studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright © 2008 John Wiley & Sons, Ltd.     

Cell encapsulation within PVA‐based hydrogels via freeze‐thawing: a one‐step scaffold formation and cell storage technique

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA‐based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue‐engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA–gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze–thaw process can be utilized to prepare and store cell‐laden hydrogels with adjustable mechanical properties. Copyright © 2009 John Wiley & Sons, Ltd.     

Microwave‐assisted fabrication of chitosan–hydroxyapatite superporous hydrogel composites as bone scaffolds

In this study, a novel scaffold fabrication method was developed by combining microwave irradiation and gas foaming. Chitosan superporous hydrogels (SPHs) and chitosan–hydroxyapatite (HA) superporous hydrogel composites (SPHCs) were prepared by using this method in the presence of crosslinking agent, glyoxal, and a gas‐blowing agent, NaHCO₃. In order to examine the effect of HA on composite structure and cellular behaviour, two types of HA particles, i.e. spherical beads in 45–80 µm diameter and powder form, were used. While rapid heating with microwave irradiation enhances gas blowing, pH increment, which is accelerated by NaHCO₃ decomposition, provides better crosslinking. Thus, interconnected and well‐established macroporous hydrogels/hydrogel composites were produced easily and rapidly (~1 min). Cell culture studies, which were carried out under static and dynamic conditions with MC3T3‐E1 pre‐osteoblastic cells, indicated that chitosan–HA bead SPHCs supported cellular proliferation and osteoblastic differentiation better than chitosan SPHs and chitosan–HA powder SPHCs. In conclusion, simultaneous gas foaming and microwave crosslinking can be evaluated for the preparation of composite scaffolds which have superior properties for bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Preparation of cell aggregates incorporating gelatin hydrogel microspheres of sugar‐responsive water solubilization

The objective of this study is to design hydrogel microspheres of a cell scaffold, which not only function as a scaffold to form cell aggregates of three‐dimensional culture but also can disappear to release growth factors in the well‐controlled manner by noncytotoxic stimulation in any timing. The hydrogel microspheres were prepared by a water‐in‐oil emulsion method from m‐aminophenylboronic acid (APBA)‐introduced gelatin (APBA–gelatin) with or without poly(vinyl alcohol) (PVA) mixing. Irrespective of the PVA concentration, the microspheres with the same diameter were prepared. The microspheres were water solubilized only by adding sorbitol of a sugar although the solubilization extent depended on the PVA concentration. When cocultured with the microspheres, mesenchymal stem cells formed cell aggregates homogeneously incorporating the microspheres. Upon adding sorbitol in the culture medium, mixed APBA–gelatin–PVA hydrogel microspheres disappeared with time in the cell aggregates. The microspheres containing basic fibroblast growth factor or bone morphogenetic protein‐2 released the respective growth factor accompanied with the microspheres disappearance. It is concluded that the present microspheres of sugar‐responsive water solubilization are promising scaffold of cell aggregates and have an ability to allow growth factors to be released in the cell aggregates when it is required.     

An additive manufacturing‐based PCL–alginate–chondrocyte bioprinted scaffold for cartilage tissue engineering

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self‐assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three‐dimensional (3D) cell‐printed scaffolds using layer‐by‐layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell‐encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell‐based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL–alginate gel constructs. PCL–alginate gels containing transforming growth factor‐β (TGFβ) showed higher ECM formation. The 3D cell‐printed scaffolds of PCL–alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL–alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell‐printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.     

Construction of engineering adipose‐like tissue in vivo utilizing human insulin gene‐modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds

We evaluated the use of a combination of human insulin gene‐modified umbilical cord mesenchymal stromal cells (hUMSCs) with silk fibroin 3D scaffolds for adipose tissue engineering. In this study hUMSCs were isolated and cultured. HUMSCs infected with Ade–insulin–EGFP were seeded in fibroin 3D scaffolds with uniform 50–60 µm pore size. Silk fibroin scaffolds with untransfected hUMSCs were used as control. They were cultured for 4 days in adipogenic medium and transplanted under the dorsal skins of female Wistar rats after the hUMSCs had been labelled with chloromethylbenzamido‐1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (CM‐Dil). Macroscopical impression, fluorescence observation, histology and SEM were used for assessment after transplantation at 8 and 12 weeks. Macroscopically, newly formed adipose tissue was observed in the experimental group and control group after 8 and 12 weeks. Fluorescence observation supported that the formed adipose tissue originated from seeded hUMSCs rather than from possible infiltrating perivascular tissue. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration in the experimental group than in the control group. SEM showed that experimental group cells had more fat‐like cells, whose volume was larger than that of the control group, and degradation of the silk fibroin scaffold was greater under SEM observation. This study provides significant evidence that hUMSCs transfected by adenovirus vector have good compatibility with silk fibroin scaffold, and adenoviral transfection of the human insulin gene can be used for the construction of tissue‐engineered adipose. Copyright © 2013 John Wiley & Sons, Ltd.     

Construction of tissue‐engineered osteochondral composites and repair of large joint defects in rabbit

In this study, a novel three‐dimensional (3D) heterogeneous/bilayered scaffold was constructed to repair large defects in rabbit joints. The scaffold includes two distinct but integrated layers corresponding to the cartilage and bone components. The upper layer consists of gelatin, chondroitin sulphate and sodium hyaluronate (GCH), and the lower layer consists of gelatin and ceramic bovine bone (GCBB). The two form a 3D bilayered scaffold (GCH–GCBB), which mimics the natural osteochondral matrix for use as a scaffold for osteochondral tissue engineering. The purpose of this study was to evaluate the efficacy of this novel scaffold, combined with chondrocytes and bone marrow stem cells (BMSCs) to repair large defects in rabbit joints. Thirty‐six large defects in rabbit femoral condyles were created; 12 defects were treated with the same scaffold combined with cells (group A); another 12 defects were treated with cell‐free scaffolds (group B); the others were untreated (group C). At 6 and 12 weeks, in group A hyaline‐like cartilage formation could be observed by histological examination; the newly formed cartilage, which stained for type II collagen, was detected by RT–PCR at high‐level expression. Most of the GCBB was replaced by bone, while little remained in the underlying cartilage. At 36 weeks, GCBB was completely resorbed and a tidemark was observed in some areas. In contrast, groups B and C showed no cartilage formation but a great amount of fibrous tissue, with only a little bone formation. In summary, this study demonstrated that a novel scaffold, comprising a top layer of GCH, having mechanical properties comparable to native cartilage, and a bottom layer composed of GCBB, could be used to repair large osteochondral defects in joints. Copyright © 2012 John Wiley & Sons, Ltd.     

Photopolymerizable chitosan–collagen hydrogels for bone tissue engineering

Photopolymerizable hydrogels derived from naturally occurring polymers have attracted significant interest in tissue‐engineering applications due to their excellent biocompatibility, hydrophilic nature favourable for cell ingrowth and ability to be cured in situ through a minimally invasive procedure. In this study, we developed a composite hydrogel consisting of photocrosslinkable methacrylated glycol chitosan (MeGC) and semi‐interpenetrating collagen (Col) with a riboflavin photoinitiator under blue light. The incorporation of Col in MeGC hydrogels enhanced the compressive modulus and slowed the degradation rate of the hydrogels. MeGC–Col composite hydrogels significantly enhanced cellular attachment, spreading, proliferation and osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) seeded on the hydrogels compared with pure MeGC hydrogels, as observed by upregulated alkaline phosphatase (ALP) activity as well as increased mineralization. Similarly, when cells were encapsulated within hydrogels, BMSCs exhibited greater proliferation, ALP activity and mineral deposits in the presence of Col. These findings demonstrate that MeGC–Col composite hydrogels may be useful in promoting bone regeneration. Copyright © 2014 John Wiley & Sons, Ltd.     

Cell–matrix and cell–cell interactions of human gingival fibroblasts on three‐dimensional nanofibrous gelatin scaffolds

An in‐depth understanding of the interactions between cells and three‐dimensional (3D) matrices (scaffolds) is pivotal to the development of novel biomaterials for tissue regeneration. However, it remains a challenge to find suitable biomimetic substrates and tools to observe cell–material and cell–cell interactions on 3D matrices. In the present study, we developed biomimetic nanofibrous 3D gelatin scaffolds (3D‐NF‐GS) and utilized confocal microscopy combined with a quantitative analysis approach to explore cell–matrix and cell–cell interactions on the 3D‐NF‐GS. Human gingival fibroblasts (HGFs) migrated throughout the 3D‐NF‐GS by 5 days and formed stable focal adhesions by 14 days. The focal adhesions were detected using integrin‐β1, phospho‐paxillin and vinculin expression, which were quantified from specific wavelength photon data generated using a spectral separation confocal microscope. As the cells became more confluent after 14 days of culture, cell–cell communication via gap junctions increased significantly. Collagen I matrix production by HGFs on 3D‐NF‐GS was visualized and quantified using a novel approach incorporating TRITC label in the scaffolds. Based on confocal microscopy, this study has developed qualitative and quantitative methods to study cell–matrix and cell–cell interactions on biomimetic 3D matrices, which provides valuable insights for the development of appropriate scaffolds for tissue regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of poly(lactide‐co‐glycolide) scaffold‐impregnated small intestinal submucosa with pores that stimulate extracellular matrix production in disc regeneration

The pore size and microstructure of scaffolds influences cell attachment, migration, proliferation and ingrowth, but the optimal pore size of scaffolds for disc tissue formation is not clearly understood. We developed porous poly(lactide‐co‐glycolide) (PLGA) scaffolds with various pore sizes for nucleus pulposus (NP) cell cultures and examined the effects of pore size on cell ingrowth and extracellular matrix (ECM) synthesis. High cell density in the small pores of scaffolds promotes collagen synthesis and cell migration through interconnected pores. Scaffolds with large pores exhibited slower cell proliferation and collagen synthesis. Guided by these results, we investigated a novel, biodegradable, synthetic/natural hybrid scaffold composed of PLGA and small intestinal submucosa (SIS) (PLGA–SIS) with the proper pore size for NP regeneration. We tested the morphological and physical properties of PLGA–SIS scaffolds and initial cell attachment and ECM production of NP in scaffolds. The mechanical and degradable properties of the PLGA–SIS scaffold were superior to those of SIS sponge and were similar to the properties of PLGA scaffold. NP cells grown on PLGA–SIS scaffold exhibited higher initial cell adhesion and ECM production than those grown on pure PLGA scaffold in a biological assay. In conclusion, this study suggests that a proper pore size of scaffolds is critical in NP regeneration, and that PLGA–SIS scaffolds with suitable pores might be useful as substrates for tissue‐engineered biodiscs. Copyright © 2012 John Wiley & Sons, Ltd.     

PDLA/PLLA and PDLA/PCL nanofibers with a chitosan‐based hydrogel in composite scaffolds for tissue engineered cartilage

Osteoarthritis (OA) is the most prevalent musculoskeletal disease in humans, causing pain, loss of joint motility and function, and severely reducing the standard of living of patients. Cartilage tissue engineering attempts to repair the damaged tissue of individuals suffering from OA by providing mechanical support to the joint as new tissue regenerates. The aim of this study was to create composite three dimensional scaffolds comprised of electrospun poly(D,L‐lactide)/poly(L‐lactide) (PDLA/PLLA) or poly(D,L‐lactide)/polycaprolactone (PDLA/PCL) with salt leached pores and an embedded chitosan hydrogel to determine the potential of these scaffolds for cartilage tissue engineering. PDLA/PLLA‐hydrogel scaffolds displayed the largest compressive moduli followed by PDLA/PCL‐hydrogel scaffolds. Dynamic mechanical tests showed that the PDLA/PLLA scaffolds had no appreciable recovery while PDLA/PCL scaffolds did exhibit some recovery. Primary canine chondrocytes produced both collagen type II and proteoglycans (primary components of extracellular matrix in cartilage) while being cultured on scaffolds composed of electrospun PDLA/PCL. As a result, a composite electrospun embedded hydrogel scaffold shows promise for treating individuals suffering from OA. Copyright © 2012 John Wiley & Sons, Ltd.     

Design and biological functionality of a novel hybrid Ti‐6Al‐4V/hydrogel system for reconstruction of bone defects

We have designed a unique injectable bioactive hydrogel comprising of alginate, gelatin, and nanocrystalline hydroxyapatite and loaded with osteoblasts, with the ability to infiltrate into three‐dimensional Ti‐6Al‐4V scaffolds with interconnected porous architecture, fabricated by electron beam melting. A two‐step crosslinking process using the EDC/NHS and CaCl₂ was adopted and found to be effective in the fabrication of cell‐loaded hydrogel/Ti‐6Al‐4V scaffold system. This hybrid Ti‐6Al‐4V scaffold/hydrogel system was designed for the reconstruction of bone defects, which are difficult to heal in the absence of suitable support materials. The hybrid Ti‐6Al‐4V/hydrogel system favourably modulated the biological functions, namely, adhesion, proliferation, cell‐to‐cell, and cell‐material communication because of the presence of extracellular matrix‐like hydrogel in the interconnected porous structure of 3D printed Ti‐6Al‐4V scaffold. The hydrogel was cytocompatible, which was proven through live/dead assay, the expression level of prominent proteins for cell adhesion and cytoskeleton, including 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay. Furthermore, the high bone formation ability of the hydrogel was confirmed using alkaline phosphatase assay. A high equilibrium water content (~97%) in the hydrogel enables the delivery of cells and bioactive molecules, necessary for bone tissue growth. Although not studied, the presence of hydrogel in the pores of the scaffold can provide the space for the cell migration as well as vascularization through it, required for the effective exchange of nutrients. In conclusion, we underscore that the 3D‐printed Ti‐6Al‐4V scaffold‐loaded with bioactive hydrogel to treat the bone defects significantly impacted cellular functions and cell‐material interaction.     

Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin–collagen hydrogel: A clinical study for diabetic wound healing

Despite the abundance of skin substitutes in the worldwide market, major hurdles in developing more complex tissues include the addition of skin appendages and vascular networks as the most important structure. The aim of this research was a clinical feasibility study of a novel prevascularized skin grafts containing the dermal and epidermal layer using the adipose stromal vascular fraction (SVF)‐derived endothelial cell population for vascular network regeneration. Herein, we characterized hydrogel with emphasis on biological compatibility and cell proliferation, migration, and vitality. The therapeutic potential of the prevascularized hydrogel transplanted on five human subjects as an intervention group with diabetic wounds was compared with nonvascularized skin grafts as the control on five patients. Wound planimetric and biometric analysis was performed using a Mann–Whitney nonparametric t‐test (p ≤ .05). The fibrin–collagen hydrogel was suitable for skin organotypic cell culture. There was a significant (p ≤ .05) increased in skin thickness and density in the vascular beds of the hypodermis measured with skin scanner compared with that in the control group. No significant macroscopic differences were observed between the intervention and control groups (p ≤ .05). In summary, we report for the first time the use of autologous dermal–epidermal skin grafts with intrinsic vascular plexus in a clinical feasibility study. The preliminary data showed that SVF‐based full‐thickness skin grafts are safe and accelerate the wound healing process. The next stage of the study is a full‐scale randomized clinical trial for the treatment of patients with chronic wounds.     

Pressure‐activated microsyringe (PAM) fabrication of bioactive glass–poly(lactic‐co‐glycolic acid) composite scaffolds for bone tissue regeneration

The aim of this work was the fabrication and characterization of bioactive glass–poly(lactic‐co‐glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA–CEL2 composite scaffolds were innovatively produced by a pressure‐activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2–PLGA composite films (CEL2 is an experimental bioactive SiO₂–P₂O₅–CaO–MgO–Na₂O–K₂O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10–50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone‐like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast‐like cells and periosteal‐derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone‐like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT–PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparation of a “Branch-Fruit” structure chitosan nanofiber physical hydrogels with high mechanical strength and pH-responsive controlled drug release properties

The poor mechanical properties of chitosan physical hydrogels seriously hinder their application in the biomedical field. Inspired by the structure of cell tissues, a novel chitosan nanofiber (CSNF)/Hyaluronic acid (HA)/β-glycerophosphate disodium (β-GP) drug-loaded hydrogel was prepared by micro-dissolution and physical crosslinking. The hydrogel has a “Branch-Fruit” structure and exhibits excellent mechanical properties, good biocompatibility and cell-adhesion properties. Human cancer cells (HeLa) can adhere to the hydrogel surface, which might facilitate tumor site-specific administration of drugs. This material also exhibits high pH sensitivity, with which drug release can be triggered under acidic conditions at pH 4.00. The mechanical strength and drug release behavior of this hydrogel can be easily adjusted by varying the CSNF content.

Representation of the gelation mechanism of CSNF/HA/β-GP precursor solution.