Bilayer Scaffold Research Articles

Fabrication and characterization of bilayer scaffolds made of decellularized dermis/nanofibrous collagen for healing of full-thickness wounds.

Skin tissue engineering has progressed from simple wound dressings to biocompatible materials with desired physico-chemical properties that can deliver regenerative biomolecules. This study describes using a novel biomimetic hybrid scaffold of decellularized dermis/collagen fibers that can continuously deliver stromal cell-derived factor-1 alpha (SDF-1α) for skin regeneration. In diabetic rat models, the idea that sustained SDF-1α infusion could increase the recruitment of CXCR4-positive cells at the injury site and improve wound regeneration was investigated. The morphology of the scaffold, its biocompatibility, and the kinetics of SDF-1 release were all assessed. SDF-1α was successfully incorporated into collagen nanofibers, resulting in a 200-h continuous release profile. The microscopic observations exhibited that cells are attached and proliferated on proposed scaffolds. As evaluated by in vivo study and histological examination, fabricated scaffold with SDF-1α release capacity exhibited a remarkably more robust ability to accelerate wound regeneration than the control group. Besides, the SDF-1α-loaded scaffold demonstrated functional effects on the proliferation and recruitment of CD31 and CXCR4-positive cells in the wound bed. Additionally, no adverse effects such as hyperplasia or scarring were found during the treatment period. It may be concluded that the fabricated hybrid scaffold based on natural polymer opens up a new option for topical administration of bioactive molecules. We believe the SDF-1α-loaded hybrid scaffold has promise for skin tissue engineering.

Functionally multifaceted scaffolds delivering bioactive compounds for treatment of infectious chronic and ischemic wounds

Chronic wounds arise due to delayed healing, disease conditions, bacterial manifestation etc. that are associated with several challenging hallmarks, mainly involving persistent inflammation, oxidative stress, infections, hypoxic conditions occurring at the wound site. Hence, for targeting such critical and multifactorial complication, an advanced combinatorial approach is prerequisite for efficient and timely healing of chronic wounds. In the present study, a versatile bilayer-type cryogel made up of chitosan–gelatin as base layer for housing the bioactive factors (nitric oxide nanoparticles as signaling molecule, cerium oxide microparticles as antioxidant, calcium peroxide microparticles as oxygen releasing moiety) has been fabricated using the cryogelation method along with a top iodine embedded antibacterial layer. The fabricated scaffold and incorporated bioactive molecules are physiochemically characterized for distinctive parameters and validated for the treatment of ischemic and infected chronic wounds model in vivo. The developed scaffolds display a high-water uptake capacity (∼93%-98%), swelling ratio (∼18.3–22.5) and more than 90% of degradation within a period of 15 days. The release kinetics reveals the release of ∼80% (within 3 days), ∼78% (within 10 days) and ∼94% (within 10 days) iodine, oxygen and nitric acid (NO), respectively. The in vitro study shows excellent biocompatibility and proliferative capacity of the scaffolds besides displaying strong antioxidant and cell migration potential in the in vitro models. The scaffold also elucidates efficacious antibacterial action against Escherichia coli and Staphylococcus aureus. Moreover, the in vivo studies demonstrate bilayer scaffold significantly expedites wound closure within a period of 21 days (∼99.6%) and regeneration in comparison to control and other groups. The histological study shows well-organized skin ultrastructure with granulation tissue, blood vessels and hair follicles in the healed wound of bilayer group. Immuno fluorescence staining results showed presence of angiogenesis marker, CD31, in the treatment groups. The results of scaffold treated ischemic wounds signify prevention of necrosis apart from maintaining native skin architecture in the scaffolds with NO and oxygen releasing properties. Thus, the combinatorial approach for chronic wounds proves effective and paves new ways in the advanced treatment strategies for complex wound conditions.

Sequential Release of Panax Notoginseng Saponins and Osteopractic Total Flavone from Poly (L-Lactic Acid) Scaffold for Treating Glucocorticoid-Associated Osteonecrosis of Femoral Head.

Glucocorticoids inhibit angiogenesis in the femoral head, which fails to nourish the bone tissue and leads to osteonecrosis. Restoring angiogenesis is not only essential for vessel formation, but also crucial for osteogenesis. Poly (L-lactic acid) (PLLA) is commonly used in the bone tissue engineering field. Panax notoginseng saponins (PNS) and osteopractic total flavone (OTF) promote angiogenesis and osteogenesis, respectively. We designed a sequentially releasing PLLA scaffold including PLLA loaded with OTF (inner layer) and PLLA loaded with PNS (outer layer). We assessed the osteogenic effect of angiogenesis in this scaffold by comparing it with the one-layered scaffold (PLLA embedded with OTF and PNS) in vivo. Results from the micro-CT showed that the data of bone mineral density (BMD), bone volume (BV), and percent bone volume (BV/TV) in the PO-PP group were significantly higher than those in the POP group (p < 0.01). Histological analyses show that the PO-PP scaffold exhibits better angiogenic and osteogenic effects compared with the one-layered scaffold. These might result from the different structures between them, where the sequential release of a bi-layer scaffold achieves the osteogenic effect of vascularization by initially releasing PNS in the outer layer. We further explored the possible mechanism by an immunohistochemistry analysis and an immunofluorescence assay. The results showed that the protein expressions of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1(CD31) in the PO-PP scaffold were significantly higher than those in the POP scaffold (p < 0.01); the protein expressions of osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) in the PO-PP scaffold were significantly higher than those in the POP scaffold (p < 0.05). Upregulating the expressions of angiogenic and osteogenic proteins might be the possible mechanism.

A 3D multifunctional bi-layer scaffold to regulate stem cell behaviors and promote osteochondral regeneration.

Osteochondral defect (OCD) regeneration remains a great challenge. Recently, multilayer scaffold simulating native osteochondral structures have aroused broad interest in osteochondral tissue engineering. Here, we developed a 3D multifunctional bi-layer scaffold composed of a kartogenin (KGN)-loaded GelMA hydrogel (GelMA/KGN) as an upper layer mimicking a cartilage-specific extracellular matrix and a hydroxyapatite (HA)-coated 3D printed polycaprolactone porous scaffold (PCL/HA) as a lower layer simulating subchondral bone. The bi-layer scaffolds were subsequently modified with tannic acid (TA) prime-coating and E7 peptide conjugation (PCL/HA-GelMA/KGN@TA/E7) to regulate endogenous stem cell behaviors and exert antioxidant activity for enhanced osteochondral regeneration. In vitro, the scaffolds could support cell attachment and proliferation, and enhance the chondrogenic and osteogenic differentiation capacity of bone marrow-derived mesenchymal stem cells (BMSCs) in a specific layer. Besides, the incorporation of TA/E7 significantly increased the biological activity of the bi-layer scaffolds including the pro-migratory effect, antioxidant activity, and the maintenance of cell viability against oxidative stress. In vivo, the developed bi-layer scaffolds enhanced the simultaneous regeneration of cartilage and subchondral bone when implanted into a rabbit OCD model through macroscopic, micro-CT, and histological evaluation. Taken together, these investigations demonstrated that the 3D multifunctional bi-layer scaffolds could provide a suitable microenvironment for endogenous stem cells, and promote in situ osteochondral regeneration, showing great potential for the clinical treatment of OCD.

Production of 3D Printed Bi-Layer and Tri-Layer Sandwich Scaffolds with Polycaprolactone and Poly (vinyl alcohol)-Metformin towards Diabetic Wound Healing.

Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by impaired insulin secretion, sensitivity, and hyperglycemia. Diabetic wounds are one of the significant complications of T2DM owing to its difficulty in normal healing, resulting in chronic wounds. In the present work, PCL/PVA, PCL/PVA/PCL, and metformin-loaded, PCL/PVA-Met and PCL/PVA-Met/PCL hybrid scaffolds with different designs were fabricated using 3D printing. The porosity and morphological analysis of 3D-printed scaffolds were performed using scanning electron microscopy (SEM). The scaffolds' average pore sizes were between 63.6 ± 4.0 and 112.9 ± 3.0 μm. Molecular and chemical interactions between polymers and the drug were investigated with Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Mechanical, thermal, and degradation analysis of the scaffolds were undertaken to investigate the physico-chemical characteristics of the scaffolds. Owing to the structure, PCL/PVA/PCL sandwich scaffolds had lower degradation rates than the bi-layer scaffolds. The drug release of the metformin-loaded scaffolds was evaluated with UV spectrometry, and the biocompatibility of the scaffolds on fibroblast cells was determined by cell culture analysis. The drug release in the PCL/PVA-Met scaffold was sustained till six days, whereas in the PCL/PVA-Met/PCL, it continued for 31 days. In the study of drug release kinetics, PCL/PVA-Met and PCL/PVA-Met/PCL scaffolds showed the highest correlation coefficients (R2) values for the first-order release model at 0.8735 and 0.889, respectively. Since the layered structures in the literature are mainly obtained with the electrospun fiber structures, these biocompatible sandwich scaffolds, produced for the first time with 3D-printing technology, may offer an alternative to existing drug delivery systems and may be a promising candidate for enhancing diabetic wound healing.

Fabrication and characterization of bilayer scaffolds - nanocellulosic cryogels - for skin tissue engineering by co-culturing of fibroblasts and keratinocytes

This study focuses on developing a microarchitectural bilayer structure for stimulating the two top layers of skin tissue (epidermis and dermis) fabricated using a one-step freeze-drying method. Cellulose nanofibers (CNFs) and poly (vinyl) alcohol (PVA) were used as a biocompatible scaffolding material, and the composition was designed in such a way that it provides physical and biological property attributes. In this work, scaffolding materials with integrated layer structures and well interconnected and open pore structures were obtained. This bilayer structure had porosity with a pore size of 19.72 μm and 90.71 μm for the simulation of the epidermis and dermis, respectively. The production and expression of laminin, collagen IV, and keratin 10 proteins in the bilayer cryogel scaffolds obtained from the immunofluorescence study were 49.7 %, 63.8 %, and 49.3 %, respectively. The extracellular matrix (ECM) was produced in each scaffold layer. The observations confirmed that the porosity and pore size of both N1 and N2 layers were appropriate for the fibroblast and keratinocyte cells, respectively. H&E stained cross-sections of bilayer cryogel scaffolds illustrated epidermal and dermal layers produced by co-culturing keratinocytes and fibroblasts. Based on the results, the bilayer CNF/PVA scaffold can be used in skin tissue engineering applications. However, more experiments and in vivo evaluations are needed to express this conclusion more accurately.

Subcutaneous osteochondral regeneration using a bilayer scaffold by leveraging angiogenic activities

• A gelatin-based bilayer scaffold with Cur/GT as upper and DS/GT as bottom layer was developed. • Cur/GT exerted antiangiogenic activity to the stable chondrogenesis of BMSCs. • DS/GT endowed proangiogenic effects to promote osteogenesis of BMSCs. • The BMSC-loaded bilayer scaffold achieved subcutaneous osteochondral regeneration. Bone marrow stem cell (BMSC) therapy is a promising method to achieve subcutaneous osteochondral regeneration. However, it remains obscure due to the incorrect angiogenic niche. Studies have demonstrated that an antiangiogenic niche promotes cartilage regeneration, whereas a proangiogenic niche facilitates bone formation. Considering that curcumin (Cur) is an antiangiogenic drug and Danshen (DS) is a proangiogenic drug, this study is the first to develop a gelatin-based bilayer porous scaffold, where curcumin-loaded gelatin (Cur/GT) was used as the upper layer and DanShen-loaded gelatin (DS/GT) was used as the bottom layer. The data revealed that both Cur and DS could be sustained and released from Cur/GT and DS/GT, respectively. When cocultured with human umbilical vein endothelial cells, Cur/GT inhibited tube formation, whereas DS/GT promoted tube formation. Furthermore, the findings demonstrated that the Cur addition in the upper layer exerted antiangiogenic activity to the stable chondrogenesis of BMSCs, the DS addition in the bottom layer endowed proangiogenic effects to promote osteogenesis of BMSCs, and the bilayer scaffold successfully achieved subcutaneous osteochondral regeneration when recolonized with BMSCs. This study provides new insight into subcutaneous osteochondral regeneration by leveraging the angiogenic microenvironment.

Mn-Doped Glass–Ceramic Bioactive (Mn-BG) Thin Film to Selectively Enhance the Bioactivity of Electrospun Fibrous Polymeric Scaffolds

In recent years, significant progress has been made in the development of new technologies to meet the demand for engineered interfaces with appropriate properties for osteochondral unit repair and regeneration. In this context, we combined two methodologies that have emerged as powerful approaches for tissue engineering application: electrospinning to fabricate a nanofibrous polymeric scaffold and pulsed laser deposition to tune and control the composition and morphology of the scaffold surface. A multi-component scaffold composed of synthetic and natural polymers was proposed to combine the biocompatibility and suitable mechanical properties of poly(D,L-lactic acid) with the hydrophilicity and cellular affinity of gelatin. As part of a biomimetic strategy for the generation of bi-functional scaffolds, we coated the electrospun fibers with a thin film of a bioactive glass–ceramic material supplemented with manganese ions. The physico-chemical properties and composition of the bi-layered scaffold were investigated, and its bioactivity, in terms of induced mineralization, was tested by incubation in a simulated body fluid buffer. The processes of the inorganic film dissolution and the calcium phosphate phases growth were followed by microscopic and spectroscopic techniques, confirming that a combination of bioactive glass–ceramics and nanofibrous scaffolds has promising potential in the regeneration of osteochondral tissue due to its ability to induce mineralization in connective tissues.

Cartilage regeneration using improved surface electrospun bilayer polycaprolactone scaffolds loaded with transforming growth factor-beta 3 and rabbit muscle-derived stem cells.

Polycaprolactone (PCL) has recently received significant attention due to its mechanical strength, low immunogenicity, elasticity, and biodegradability. Therefore, it is perfectly suitable for cartilage tissue engineering. PCL is relatively hydrophobic in nature, so its hydrophilicity needs to be enhanced before its use in scaffolding. In our study, first, we aimed to improve the hydrophilicity properties after the network of the bilayer scaffold was formed by electrospinning. Electrospun bilayer PCL scaffolds were treated with ozone and further loaded with transforming growth factor-beta 3 (TGFβ3). In vitro studies were performed to determine the rabbit muscle-derived stem cells’ (rMDSCs) potential to differentiate into chondrocytes after the cells were seeded onto the scaffolds. Statistically significant results indicated that ozonated (O) scaffolds create a better environment for rMDSCs because collagen-II (Coll2) concentrations at day 21 were higher than non-ozonated (NO) scaffolds. In in vivo studies, we aimed to determine the cartilage regeneration outcomes by macroscopical and microscopical/histological evaluations at 3- and 6-month time-points. The Oswestry Arthroscopy Score (OAS) was the highest at both mentioned time-points using the scaffold loaded with TGFβ3 and rMDSCs. Evaluation of cartilage electromechanical quantitative parameters (QPs) showed significantly better results in cell-treated scaffolds at both 3 and 6 months. Safranin O staining indicated similar results as in macroscopical evaluations—cell-treated scaffolds revealed greater staining with safranin, although an empty defect also showed better results than non-cell-treated scaffolds. The scaffold with chondrocytes represented the best score when the scaffolds were evaluated with the Mankin histological grading scale. However, as in previous in vivo evaluations, cell-treated scaffolds showed better results than non-cell-treated scaffolds. In conclusion, we have investigated that an ozone-treated scaffold containing TGFβ3 with rMDSC is a proper combination and could be a promising scaffold for cartilage regeneration.

Biocompatible organic electrochemical transistor on polymeric scaffold for wound healing monitoring

Nowadays, no wearable device is available to continuously monitor the ulcer status. The literature reports a pH range between 6.5 and 9 for chronic or infected wounds. This study aims to produce an innovative scaffold able to monitoring wound pH and therefore ulcers healing. The scaffold was manufactured by spray, phase-inversion technique using a synthetic biocompatible material, poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS). A bi-layer micro-fibrillar tubular scaffold was obtained using: (a) 2% polymer solution and H2O as non-solvent; (b) 0.2% and H2O. Poly(3,4‐ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) is a biocompatible conductive polymer used as active material in a biosensor due to high sensitivity to positive ions in liquid environment allowing to determine ions concentration in easy and stable way. The biocompatible scaffold was functionalized by inkjet-printing of a PEDOT:PSS to develop organic electrochemical transistor (OECT) architecture. The electrochemical device was connected with electric contacts to create a channel and a gate electrode to control the modulation changes of the sensor. The morphological analysis of PEtU-PDMS scaffold surfaces was performed using a stereo-microscope after Sudan Black B staining while the PEDOT:PSS pattern by SEM microscopy. The device functionality was proved on human serum at different pH (between four and ten). The morphological analysis showed a dense, non-porous surface obtained with the 2% solution, while a porous surface was obtained with the 0.2% solution where the PEDOT:PSS was positioned. This feature, maintained after lyophilization and re-hydratation, enables a thin PEDOT:PSS film deposition in a continuous and homogeneus pattern. The characteristic of the device was tested showing the sensitivity to saline concentration and the effective functionality of the device. Moreover, the device response shows a dependence to pH variations as well as the transconductance. Obtained data suggest that the proposed sensorised scaffold could be used as a wearable detector for wound healing monitoring in patients affected by chronic lesions.

Single unit functionally graded bioresorbable electrospun scaffold for scar-free full-thickness skin wound healing.

Full-thickness wounds are difficult to heal spontaneously. Scaffolds, meant for treating full-thickness wounds, should ensure proper tissue regeneration, both structurally and functionally. An ideal scaffold should mimic the physical, mechanical and biochemical properties of natural skin. However, available mono- or bi-layer skin scaffolds lack in the precise architecture and functionality, thus, failing to provide scar-free regeneration of full-thickness skin wounds. These unmet challenges of scar-free skin regeneration have been addressed in the present study for the first time. This research deals with the synthesis of a low-cost, structurally and functionally graded single unit biodegradable polymeric scaffold. The functional gradient in this scaffold was achieved by varying polymer concentration and electrospinning parameters. This gradient in the scaffold provided the required microenvironment for proper functional and structural reconstruction of all the layers of natural skin. The mechanical property of the scaffold matched that of the natural skin. Besides, the degradation kinetics of the scaffold was in coordination with the regeneration time for the full-thickness wound. The porosity and hydrophilicity gradients of the scaffold helped it mimic the in vivo hypodermal, dermal and epidermal microenvironments of the skin, simultaneously. Co-culturing PCS-201 (dermal fibroblasts) and HaCaT (keratinocytes) on the scaffold resulted in successful regeneration through cellular proliferation, differentiation and organization of the skin tissue. The scaffold also displayed better wound healing in vivo, in terms of speedy wound closure and proper tissue regeneration, in comparison to the standard treatment. Altogether, this study successfully established a simple, one-step synthesis process of a functionally graded, bioresorbable scaffold for scar-free, native-like, structural and functional regeneration of full-thickness skin wounds. Due to cost-effectiveness, easy synthesis process and microarchitectural features, the designed scaffold possesses a potential of translation to a good commercial wound healing product.

A bilayer collagen scaffold with bevacizumab stabilizes chondrogenesis and promotes osteochondral regeneration

Stem cell-based tissue engineering represents a promising therapeutic for osteochondral restoration. A bilayer scaffold with an appropriate niche can simultaneously induce stem cells for cartilage and bone regeneration. However, the engineered cartilage derived from stem cells tends to ossify due to vascular invasion. Bevacizumab possesses pronounced anti-angiogenic activity by inhibiting the vascular endothelial growth factor (VEGF) signaling pathway. Here, we prepared a bilayered collagen-based scaffold with bevacizumab loaded in the upper layer (Bev/collagen) to promote stable osteochondral regeneration. We show that this Bev/collagen scaffold possesses suitable bevacizumab release kinetics (nearly 100% at 7 weeks) to achieve the anti-angiogenic activity and prevents vascular invasion. When colonized with bone marrow stem cells, it also exhibited cytocompatibility to support in vitro cartilage regeneration. The engineered cartilage successfully maintained cartilaginous property in the upper layer after subcutaneous implantation in nude mice for 4 weeks and in situ transplantation in a rabbit osteochondral defect model for 8 weeks, resulting in satisfactory osteochondral regeneration. This study highlights a new avenue for osteochondral regeneration by leveraging anti-angiogenesis activity.

Bilayer silk fibroin/sodium alginate scaffold promotes vascularization and advances inflammation stage in full-thickness wound

An ideal wound dressing for full-thickness wound regeneration should offer desirable biocompatibility, adequate mechanical properties, barrier function, and cellular regulation. Here, a bilayer scaffold resembling the hierarchical structure of human skin was developed using silk fibroin and sodium alginate. The upper membrane was prepared through casting and functioned as the epidermis, whereas the lower porous scaffold was prepared by freeze-drying and mimicked extracellular matrix structures. The membrane had nonporous structure, desirable mechanical properties, moderate hydrophilic surface, and suitable water vapor transmission rate, whereas the porous scaffold revealed 157.61 ± 41.67 µm pore size, 86.10 ± 3.60% porosity, and capability of stimulating fibroblast proliferation. The combination of the two structures reinforced the tensile strength by five-fold and provided protection from wound dehydration. A suitable degradation rate reduced potential administration frequency. Furthermore, an in vivo rabbit full-thickness wound healing test demonstrated that the bilayer scaffold facilitated wound closure, granulation tissue formation, re-epithelialization and skin component transition towards normal skin by providing a moist wound environment, advancing the inflammation stage, and stimulating angiogenesis. Collectively, as an off-the-shelf and cell-free wound dressing with single topical administration, the bilayer scaffold is a promising wound dressing for full-thickness wound regeneration.

Study on bioactive PEGDA/ECM hybrid bi-layered hydrogel scaffolds fabricated by electro-writing for cartilage regeneration

Articular cartilage injury is a common disease in orthopedics. Because of the poor repair ability of articular cartilage itself, medical intervention is often required for articular cartilage injury. A promising tool for cartilage injury repair is electro-writing (EW), a 3D printing technology, which can be used to prepare high-precision and high-porosity layered active scaffolds, thereby allowing it to imitate the layered structure of cartilage. In this study, we developed a hybrid ink with good EW printability and formability based on polyethylene glycol diacrylate (PEGDA) and extracellular matrix, in which cells can be mixed for printing. Moreover, growth factors were also integrated into the printed material, such that the upper layer of the scaffold was loaded with transforming growth factor-β1 (TGF-β) + bone morphogenetic protein-7 (BMP-7) and the lower layer was loaded with TGF-β+insulin growth factor-1 (IGF-1). The whole scaffold encompassed layered active ingredients and can functionally repair damaged cartilage after being implanted in the body. In summary, this study explored the printability and formability of EW hydrogel inks, based on which an original hybrid cell hydrogel ink was developed for constructing a bioactive cartilage repair scaffold capable of inducing cell differentiation to promote cartilage repair.

Fabrication of vitamin K3-carnosine peptide-loaded spun silk fibroin fibers/collagen bi-layered architecture for bronchopleural fistula tissue repair and regeneration applications.

Bronchial and pleural injuries with persistent air leak pose a threat in the repair and regeneration of pulmonary diseases. The need to arrive at a highly efficient therapy for closure of bronchopleural fistula (BPF) so as to effectively suppress inflammation, infection and repair the damaged pleural space caused by cancer as well as contractile restoration of bronchopleural scars remain a significant clinical challenge. Herein, we have designed and developed potent bioactive vitamin K3 carnosine peptide (VKC)-loaded spun SF fibroin fibers/collagen bi-layered 3D scaffold for bronchopleural fistula tissue engineering applications. The VKC drug showed excellent cell viability in human bronchial epithelial cells (HBECs), in addition to its pronounced higher cytotoxicity against the A549 lung cancer cell line with an IC50 of 5μg/mL. Furthermore, VKC displayed a strong affinity with the catalytic site of EGFR (PDB ID: 1M17) and VEGFR2 (PDB ID: 4AGD, 4ASD) receptors in molecular docking studies. Following which the spun SF-VKC (primary layer) and collagen film (top layer) constructed bi-layered CSVKC were structurally elucidated and its morphological, physicochemical and biological characterizations were well examined. The bi-layered scaffold showed superior biocompatibility and cell migration ability in HBECs than other scaffolds. Interestingly, the CSVKC revealed rapid HBECs motility towards scratched regions for fast healing in vitro bronchial tissue engineering. In vivo biocompatibility and angiogenesis studies of the prepared scaffolds were evaluated and the results obtained demonstrated excellent new tissue formation and neovascularization in the bi-layered architecture rather than others. Therefore, our results suggest that the potent antibacterial and anticancer therapeutic agent (VKC)-impregnated silk fibroin fibers/collagen bi-layered 3D biomaterial could be useful in treating cancerous BPF and pulmonary diseases in future.

Dual-Layered Approach of Ovine Collagen-Gelatin/Cellulose Hybrid Biomatrix Containing Graphene Oxide-Silver Nanoparticles for Cutaneous Wound Healing: Fabrication, Physicochemical, Cytotoxicity and Antibacterial Characterisation.

Tissue engineering products have grown rapidly as an alternative solution available for chronic wound and burn treatment. However, some drawbacks include additional procedures and a lack of antibacterial properties that can impair wound healing, which are issues that need to be tackled effectively for better wound recovery. This study aimed to develop a functionalized dual-layered hybrid biomatrix composed of collagen sponge (bottom layer) to facilitate cell proliferation and adhesion and gelatin/cellulose hydrogel (outer layer) incorporated with graphene oxide and silver nanoparticles (GC-GO/AgNP) to prevent possible external infections post-implantation. The bilayer hybrid scaffold was crosslinked with 0.1% (w/v) genipin for 6 h followed by advanced freeze-drying technology. Various characterisation parameters were employed to investigate the microstructure, biodegradability, surface wettability, nanoparticles antibacterial activity, mechanical strength, and biocompatibility of the bilayer bioscaffold towards human skin cells. The bilayer bioscaffold exhibited favourable results for wound healing applications as it demonstrated good water uptake (1702.12 ± 161.11%), slow rate of biodegradation (0.13 ± 0.12 mg/h), and reasonable water vapour transmission rate (800.00 ± 65.85 gm−2 h−1) due to its porosity (84.83 ± 4.48%). The biomatrix was also found to possess hydrophobic properties (48.97 ± 3.68°), ideal for cell attachment and high mechanical strength. Moreover, the hybrid GO-AgNP promoted antibacterial properties via the disk diffusion method. Finally, biomatrix unravelled good cellular compatibility with human dermal fibroblasts (>90%). Therefore, the fabricated bilayer scaffold could be a potential candidate for skin wound healing application.

Design and fabrication method of bi-layered fibrous scaffold for cartilage regeneration

An advanced therapy construct for regeneration of articular cartilage is reported which was fabricated by cryo-electrospinning as 3D multi-layered poly[e]caprolactone matrix harbouring chondrogenic growth factor TGFβ-3. The layers have been designed to favour the cartilage regeneration (chondral layer) and bone tissue formation (subchondral layer) by additives of regenerated cellulose and hydroxyapatite, respectively. The overall porosity of the chondral layer was 90.7% and the subchondral layer – 94.4% with a pore diameter ranges of 50–300 µm and 75–400 µm respectively but maintaining a low fibre diameter of 9.3 ± 4.1 µm and 9.1 ± 2.1 µm. The hydrophilicity was enhanced and functional groups essential for growth factor incorporation were introduced by surface ozone treatment. The scaffold was characterised by SEM, µCT, FTIR, WCA, XRD, TGA, and DSC techniques. The processing of scaffold by chemical modifications resulted in the increase of biodegradability of the scaffold, as indicated by the decrease of the crystallinity, thermal stability, melting peak, and hydrophobicity, but maintaining its sufficient mechanical properties (Young’s modulus equalled to 1.8 ± 0.1 MPa for chondral layer and 1.1 ± 0.2 MPa for subchondral). The release kinetics of TGFβ-3 followed the Korsmeyer–Peppas model, reaching 39.3% after 72 h, and the estimated 50% release after 155 h.

A composite bilayer scaffold functionalized for osteochondral tissue regeneration in rat animal model.

Osteochondral defects are defined most typically by damages to both cartilage and subchondral bone tissue. It is challenging to develop bilayered scaffolds that regenerate both of these lineages simultaneously. In the present study, an electrospun bilayer nanofibrous scaffold was designed to repair osteochondral lesions. A nanocomposite of hydroxyapatite, strontium, and reduced graphene oxide were combined with polycaprolactone polymer to fabricate the osteogenic differentiation layer. Additionally, the chondrogenic differentiation layer was also formed using polyethersulfone polymer and benzyl hyaluronan. The physical, mechanical, and chemical properties of the scaffolds were determined, and adipose-derived mesenchymal stem cells were cultured on each layer to evaluate their biocompatibility and differentiation potential. Cell viability, mineralization, alkaline phosphatase enzyme (ALP) expression, and extracellular calcium deposition were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, alizarin red staining, ALP activity, and calcium deposition. Real-time polymerase chain reaction (PCR) was used to assess the expression levels of osteogenic (Collagen I, Runx II, ALP, Osteocalcin) and chondrogenic (Sox9, Collagen II (Col II), Aggrecan) genes. Finally, the osteochondral scaffold was created by electrospinning these two layers for 2days. The scaffold was grafted into the osteochondral defect of a Wistar rat's knee. 60days after surgery, real-time PCR, immunohistochemistry (IHC), and hematoxylin and eosin staining were performed. The expression of chondrogenic and osteogenic genes was increased compared to the control group, as confirmed by real-time PCR. Furthermore, IHC revealed a rise in Col II and Collagen X expression. Finally, in vivo and in vitro studies have shown that the electrospun bilayer scaffold is biocompatible, which facilitates osteochondral healing.

Hyaluronic acid-based biphasic scaffold with layer-specific induction capacity for osteochondral defect regeneration

The repair of cartilage and subchondral bone defects, especially the former, remains a challenge in clinical practice. An increasing number of studies have shown that the use of biomaterials has great advantages in regulating tissue repair, with lower cost and risk than the use of cytokines or cells. However, great challenges remain because of the marked differences between cartilage and subchondral bone. Here, biphasic osteochondral scaffolds with layer-specific differentiation were designed and developed. Hyaluronic acid (HA) was used as the matrix in the upper and lower layers, and differences in physicochemical properties and biological properties between the two layers were realized by different chemical crosslinking methods. The cartilage regeneration layer exhibited stress relaxation from the HA hydrazone crosslinking dynamics. Additionally, a porous HA hydrogel with dominant elasticity was prepared for subchondral bone repair. Importantly, a 3D-printed scaffold bracket could keep close integration of two layers and provided mechanical support, aiding the long-term tissue regeneration process. The in vitro and in vivo results indicated that the HA-base bilayer scaffold consisting of a dynamic HA layer and a porous HA layer realized the simultaneous integrated regeneration of articular cartilage and subchondral bone. Such scaffolds represent potential new materials for osteochondral regeneration.

Bilayered extracellular matrix derived scaffolds with anisotropic pore architecture guide tissue organization during osteochondral defect repair

While some clinical advances in cartilage repair have occurred, osteochondral (OC) defect repair remains a significant challenge, with current scaffold-based approaches failing to recapitulate the complex, hierarchical structure of native articular cartilage (AC). To address this need, we fabricated bilayered extracellular matrix (ECM)-derived scaffolds with aligned pore architectures. By modifying the freeze-drying kinetics and controlling the direction of heat transfer during freezing, it was possible to produce anisotropic scaffolds with larger pores which supported homogenous cellular infiltration and improved sulfated glycosaminoglycan deposition. Neo-tissue organization in vitro could also be controlled by altering scaffold pore architecture, with collagen fibres aligning parallel to the long-axis of the pores within scaffolds containing aligned pore networks. Furthermore, we used in vitro and in vivo assays to demonstrate that AC and bone ECM derived scaffolds could preferentially direct the differentiation of mesenchymal stromal cells (MSCs) towards either a chondrogenic or osteogenic lineage respectively, enabling the development of bilayered ECM scaffolds capable of spatially supporting unique tissue phenotypes. Finally, we implanted these scaffolds into a large animal model of OC defect repair. After 6 months in vivo, scaffold implantation was found to improve cartilage matrix deposition, with collagen fibres preferentially aligning parallel to the long axis of the scaffold pores, resulting in a repair tissue that structurally and compositionally was more hyaline-like in nature. These results demonstrate how scaffold architecture and composition can be spatially modulated to direct the regeneration of complex interfaces such as the osteochondral unit, enabling their use as cell-free, off-the-shelf implants for joint regeneration. Statement of significanceThe architecture of the extracellular matrix, while integral to tissue function, is often neglected in the design and evaluation of regenerative biomaterials. In this study we developed a bilayered scaffold for osteochondral defect repair consisting of tissue-specific extracellular matrix (ECM)-derived biomaterials to spatially direct stem/progenitor cell differentiation, with a tailored pore microarchitecture to promote the development of a repair tissue that recapitulates the hierarchical structure of native AC. The use of this bilayered scaffold resulted in improved tissue repair outcomes in a large animal model, specifically the ability to guide neo-tissue organization and therefore recapitulate key aspects of the zonal structure of native articular cartilage. These bilayer scaffolds have the potential to become a new therapeutic option for osteochondral defect repair.