Injectable Scaffold Research Articles

Accelerated bony defect healing based on chitosan thermosensitive hydrogel scaffolds embedded with chitosan nanoparticles for the delivery of BMP2 plasmid DNA.

Regeneration of alveolar bone is among the main purposes of periodontal treatment. However, it is a challenge for both dentists and patients. An injectable chitosan-based thermosensitive hydrogel scaffold (CS/CSn-GP) was successfully designed and prepared with the incorporation of bone morphogenetic protein-2 plasmid DNA (pDNA-BMP2)-loaded chitosan nanoparticles (CS/CSn(pDNA-BMP2)-GP) in this study. The aim of this study was to evaluate the regenerative effect of this scaffold on the endogenous repair of alveolar bone. Thermosensitive properties, SEM scans and in vivo inflammatory reactions and degradation were evaluated. The CS/CSn-GP solution was initially injected into the muscle pouches of rats, where it changed into a gel, resulting in non-specific inflammation. It effectively enhanced new bone formation in rat calvarial defects and enhanced bony defect healing in beagle dogs. All results indicated that the CS/CSn(pDNA-BMP2)-GP complex system is a potential candidate as a targeted, multi-effect scaffold for the endogenous repair of alveolar bone. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 265-273, 2017.

Covalent and injectable chitosan-chondroitin sulfate hydrogels embedded with chitosan microspheres for drug delivery and tissue engineering

Injectable hydrogels and microspheres derived from natural polysaccharides have been extensively investigated as drug delivery systems and cell scaffolds. In this study, we report a preparation of covalent hydrogels basing polysaccharides via the Schiff' base reaction. Water soluble carboxymethyl chitosan (CMC) and oxidized chondroitin sulfate (OCS) were prepared for cross-linking of hydrogels. The mechanism of cross-linking is attributed to the Schiff' base reaction between amino and aldehyde groups of polysaccharides. Furthermore, bovine serum albumin (BSA) loaded chitosan-based microspheres (CMs) with a diameter of 3.8–61.6μm were fabricated by an emulsion cross-linking method, followed by embedding into CMC-OCS hydrogels to produce a composite CMs/gel scaffold. In the current work, gelation rate, morphology, mechanical properties, swelling ratio, in vitro degradation and BSA release of the CMs/gel scaffolds were examined. The results show that mechanical and bioactive properties of gel scaffolds can be significantly improved by embedding CMs. The solid CMs can serve as a filler to toughen the soft CMC-OCS hydrogels. Compressive modulus of composite gel scaffolds containing 20mg/ml of microspheres was 13KPa, which was higher than the control hydrogel without CMs. Cumulative release of BSA during 2weeks from CMs embedded hydrogel was 30%, which was significantly lower than those of CMs and hydrogels. Moreover, the composite CMs/gel scaffolds exhibited lower swelling ratio and slower degradation rate than the control hydrogel without CMs. The potential of the composite hydrogel as an injectable scaffold was demonstrated by encapsulation of bovine articular chondrocytes in vitro. These results demonstrate the potential of CMs embedded CMC-OCS hydrogels as an injectable drug and cell delivery system in cartilage tissue engineering.

Biopolymeric hydrogels − nanostructured TiO2 hybrid materials as potential injectable scaffolds for bone regeneration

The present work aims at development of novel hybrid materials from genipin crosslinked collagen or collagen/chitosan hydrogels containing various types of TiO2 nanoparticles characterized with different anatase/rutile ratios. Collagen and chitosan were selected as hydrogel components since they are biopolymers being, like collagen, the major compound present in extracellular matrix or exhibit structural similarity to glycosaminoglycans, like chitosan. TiO2 nanoparticles were introduced to the hydrogel matrices to improve their mechanical properties as well as bioactivity. A series of twelve novel hybrid materials were prepared and their physicochemical, mechanical and biological properties were evaluated.It was found that TiO2 nanostructures introduced to the hydrogels have significant influence on the swelling properties of the synthesized hybrids and their impact is strongly dependent on the type of matrices. The surfaces of hybrid materials were found to be more hydrophilic than these of corresponding hydrogel matrix. It was also observed that, the storage modulus values of the hybrids based on collagen-chitosan hydrogel are comparable to these for plain hydrogels what indicates that the mechanical properties of the materials obtained are satisfactory for possible biomedical application. The in vitro cell culture studies have shown that prepared materials are biocompatible as they can support mitochondrial activity of MEFs as well as MG-63 cells. In vitro experiments performed under simulated body fluid (SBF) conditions have revealed that all studied TiO2 nanoparticles present in hydrogel matrices, regardless of anatase/rutile ratio, successfully induced formation of apatite-like structures. The hybrid materials developed here are promising candidates for preparation of bioactive, injectable scaffolds for tissue engineering.

An injectable acellular matrix scaffold with absorbable permeable nanoparticles improves the therapeutic effects of docetaxel on glioblastoma.

Intratumoral drug delivery (IT) is an inherently appealing approach for concentrating toxic chemotherapies at the site of action. However, for most chemotherapies, poor tumor penetration and short retention at the administration site limit their anti-tumor effects. In this work, we describe permeable nanoparticles (NPs) prepared with a novel amphiphilic polymer, RRR-α-tocopheryl succinate-grafted-ε-polylysine conjugate (VES-g-ε-PLL). The nanoparticles (NPs) of VES-g-ε-PLL exhibited an ultra-small hydrodynamic diameter (20.8nm) and positive zeta potential (20.6mV), which facilitate strong glioma spheroid penetration ability invitro. Additionally, the hydrophobic model drug docetaxel (DTX) could be effectively encapsulated in the nanoparticles with 3.99% drug loading and 73.37% encapsulation efficiency. To prolong the retention time of DTX-loaded nanoparticles (DTX-NPs) in the tumor, intact decellularized brain extracellular matrix (dBECM) derived from healthy rats was used as a drug depot to adsorb the ultra-small DTX-NPs. The intact DTX-NPs-adsorbing dBECM scaffold was further homogenized into an injectable DTX-NPs-dBECM suspension for intratumoral administration. The DTX-NPs-dBECM suspension exhibited slower DTX release than naked DTX-NPs without compromising the tumor penetration ability of DTX-NPs. An antitumor study showed that the DTX-NPs-dBECM suspension exhibited more powerful invitro inhibition of tumor spheroid growth than free DTX solution or DTX-NPs. Due to strong tumor penetration ability and prolonged retention, DTX-NPs-dBECM led to complete suppression of glioma growth invivo at 28 days after treatment. The therapeutic mechanism was due to enhanced proliferation inhibition and apoptosis of tumor cells and angiogenesis inhibition of glioma after treatment with DTX-NPs-dBECM. Finally, the safety of DTX-NPs-dBECM at the therapeutic dose was demonstrated via pathological HE assay from heart, liver, spleen, lung and kidney tissues. In conclusion, permeable nanoparticle-absorbing dBECM is a potential carrier for intratumoral delivery of common chemotherapeutics.

Therapeutic Effect of Neurotrophin-3 Treatment in an Injectable Collagen Scaffold Following Rat Spinal Cord Hemisection Injury.

Spinal cord injury (SCI) patients display varying quantities of spinal cord tissue damage with injuries that present as complete, incomplete or compressive. One theory proposed to repair the injured spinal cord and regain motor control is to regenerate axons through the lesion site. This study was designed to quantify the impact of a local injectable in situ forming hydrogel reservoir therapy following rat hemisection SCI. We investigated the effect of hydrogel only treatment following SCI in addition to hydrogels loaded with a neurotrophic factor, Neurotrophin-3 (NT-3), immediately following SCI. Functional recovery, assessed by Basso Beattie Bresnahan (BBB) locomotor test, and local healing mechanisms, including neuronal growth, glial scar formation, inflammation and collagen deposition were investigated one and 6 weeks postsurgery. Delivery of an injectable hydrogel significantly increased functional recovery at four and 6 weeks post injury. In addition, a significant reduction in the inhibitory glial scar and in inflammation was observed at the injury site. Similarly hydrogel + NT-3 delivered directly into the injury site significantly reduced glial scarring and collagen deposition. The hydrogel + NT-3 also resulted in a significant increase in neurons at 6 weeks post injury. This study represents a novel and effective therapy combining growth factor and a biomaterial based therapy following SCI.

Extremely Stable Supramolecular Hydrogels Assembled from Nonionic Peptide Amphiphiles.

Peptide hydrogels with high stability in different media are of great interest in biomedical applications. In this paper, we report an easy, fast, and scalable method for preparing a family of nonionic peptide amphiphiles (PAs) obtained by direct aminolysis of alkyl-oilgo(γ-benzyl-l-glutamate) samples, which were synthesized via the alkyl amine-initiated sequence ring-opening reaction of α-amino acid N-carboxyanhydrides. One great advantage of this method is that vast chemical diversity and large-scale yields can be achieved easily using commercially available hydramines. These PA samples can readily form a clear hydrogel without any external aid and show exceptionally enhanced gelation properties with a critical gelation concentration as low as 0.05 wt %. The hydrogels are highly stable against extreme pH values of 1 and 14 and a high salt concentration of 200 mM NaCl. These properties combined with the shear-thinning properties make these PA hydrogels ideal candidates for the new generation of injectable scaffolds.

Injectable and microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels

In this work, an emulsion crosslinking method was developed to produce chitosan-genipin microgels which acted as an injectable and microporous scaffold. Chitosan was characterized with respect to pH by light scattering and aqueous titration. Microgels were characterized with swelling, light scattering, and rheometry of densely-packed microgel solutions. The results suggest that as chitosan becomes increasingly deprotonated above the pKa, repulsive forces diminish and intermolecular attractions cause pH-responsive chain aggregation; leading to microgel–microgel aggregation as well. The microgels with the most chitosan and least cross-linker showed the highest yield stress and a storage modulus of 16kPa when condensed as a microgel paste at pH 7.4. Two oppositely-charged growth factors could be encapsulated into the microgels and endothelial cells were able to proliferate into the 3D microgel scaffold. This work motivates further research on the applications of the chitosan microgel scaffold as an injectable and microporous scaffold in regenerative medicine.

Self-Healing Elastin-Bioglass Hydrogels.

Tailorable hydrogels that are mechanically robust, injectable, and self-healable, are useful for many biomedical applications including tissue repair and drug delivery. Here we use biological and chemical engineering approaches to develop a novel in situ forming organic/inorganic composite hydrogel with dynamic aldimine cross-links using elastin-like polypeptides (ELP) and bioglass (BG). The resulting ELP/BG biocomposites exhibit tunable gelling behavior and mechanical characteristics in a composition and concentration dependent manner. We also demonstrate self-healing in the ELP/BG hydrogels by successfully reattaching severed pieces as well as through rheology. In addition, we show the strength of genetic engineering to easily customize ELP by fusing cell-stimulating "RGD" peptide motifs. We showed that the resulting composite materials are cytocompatible as they support the cellular growth and attachment. Our robust in situ forming ELP/BG composite hydrogels will be useful as injectable scaffolds for delivering cell and drug molecules to promote soft tissue regeneration in the future.

Hydroxyapatite nanoparticle injectable hydrogel scaffold to support osteogenic differentiation of human mesenchymal stem cells.

Bone loss associated with degenerative disease and trauma is a clinical problem increasing with the aging population. Thus, effective bone augmentation strategies are required; however, many have the disadvantages that they require invasive surgery and often the addition of expensive growth factors to induce osteoblast differentiation. Here, we investigated a LaponiteÒ crosslinked, pNIPAM-DMAc copolymer (L-pNIPAM-co-DMAc) hydrogel with hydroxyapatite nanoparticles (HAPna), which can be maintained as a liquid ex vivo, injected via narrow-gauge needle into affected bone, followed by in situ gelation to deliver and induce osteogenic differentiation of human mesenchymal stem cells (hMSC). L-pNIPAM-co-DMAc hydrogels were synthesised and HAPna added post polymerisation. Commercial hMSCs from one donor (Lonza) were incorporated in liquid hydrogel, the mixture solidified and cultured for up to 6 weeks. Viability of hMSCs was maintained within hydrogel constructs containing 0.5mg/mL HAPna. SEM analysis demonstrated matrix deposition in cellular hydrogels which were absent in acellular controls. A significant increase in storage modulus (G') was observed in cellular hydrogels with 0.5mg/mL HAPna. Semi-quantitative immunohistochemistry and histological analysis demonstrated that bone differentiation markers and collagen deposition was induced within 48h, with increased calcium deposition with time. The thermally triggered hydrogel system, described here, was sufficient without the need of additional growth factors or osteogenic media to induce osteogenic differentiation of commercial hMSCs. Preliminary data presented here will be expanded on multiple patient samples to ensure differentiation is seen in these samples. This system could potentially reduce treatment costs and simplify the treatment strategy for orthopaedic repair and regeneration.

Biodegradable thermogelling polymers for biomedical applications

Abstract

Injectable porous nano-hydroxyapatite/chitosan/tripolyphosphate scaffolds with improved compressive strength for bone regeneration

In this study we have fabricated porous injectable spherical scaffolds using chitosan biopolymer, sodium tripolyphosphate (TPP) and nano-hydroxyapatite (nHA). TPP was primarily used as an ionic crosslinker to crosslink nHA/chitosan droplets. We hypothesized that incorporating nHA into chitosan could support osteoconduction by emulating the mineralized cortical bone structure, and improve the Ultimate Compressive Strength (UCS) of the scaffolds. We prepared chitosan solutions with 0.5%, 1% and 2% (w/v) nHA concentration and used simple coacervation and lyophilization techniques to obtain spherical scaffolds. Lyophilized spherical scaffolds had a mean diameter of 1.33mm (n=25). Further, portion from each group lyophilized scaffolds were soaked and dried to obtain Lyophilized Soaked and Dried (LSD) scaffolds. LSD scaffolds had a mean diameter of 0.93mm (n=25) which is promising property for the injectability. Scanning Electron Microscopy images showed porous surface morphology and interconnected pore structures inside the scaffolds. Lyophilized and LSD scaffolds had surface pores <10 and 2μm, respectively. 2% nHA/chitosan LSD scaffolds exhibited UCS of 8.59MPa compared to UCS of 2% nHA/chitosan lyophilized scaffolds at 3.93MPa. Standardize UCS values were 79.98MPa and 357MPa for 2% nHA/chitosan lyophilized and LSD particles respectively. One-way ANOVA results showed a significant increase (p<0.001) in UCS of 1% and 2% nHA/chitosan lyophilized scaffolds compared to 0% and 0.5% nHA/chitosan lyophilized scaffolds. Moreover, 2% nHA LSD scaffolds had significantly increased (p<0.005) their mean UCS by 120% compared to 2% nHA lyophilized scaffolds. In a drawback, all scaffolds have lost their mechanical properties by 95% on the 2nd day when fully immersed in phosphate buffered saline. Additionally live and dead cell assay showed no cytotoxicity and excellent osteoblast attachment to both lyophilized and LSD scaffolds at the end of 14th day of in vitro studies. 2% nHA/chitosan scaffolds showed higher osteoblast attachment than 0% nHA/chitosan scaffolds.

Injectable hydrogel-based scaffolds for tissue engineering applications

Abstract Hydrogels are highly hydrated materials that may absorb from 10% to 20% up to hundreds of times their dry weight in water and are composed of three-dimensional hydrophilic polymeric networks that are similar to those in natural tissue. The structural integrity of hydrogels depends on cross-links formed between the polymer chains. Hydrogels have been extensively explored as injectable cell delivery systems, owing to their high tissue-like water content, ability to mimic extracellular matrix, homogeneously encapsulated cells, efficient mass transfer, amenability to chemical and physical modifications, and minimally invasive delivery. A variety of naturally and synthetically derived materials have been used to form injectable hydrogels for tissue engineering applications. The current review article focuses on these biomaterials, on the design parameters of injectable scaffolds, and on the in situ gelling of their hydrogel systems. The last section of this article describes specific examples of catheter-based delivery systems.

Formulation and in vitro characterization of a composite biodegradable scaffold as antibiotic delivery system and regenerative device for bone

A biodegradable composite scaffold, useful as tridimensional bone regenerative scaffold (3D) and antibiotic delivery system (DDs), is here formulated and characterized. The device, called as 3D-DDS, is an injectable or moldable scaffold based on chitosan chloride crosslinked with glycerol 2-phosphate disodium salt and laden with bovine bone substitute (BBs) granules and gentamicin loaded polylactide-co-glycolide-co-poly ethilenglycol (PLGA-PEG) microparticles. 3D-DDS characterization results show that microparticles are homogeneously distributed in the composite matrix, and do not interfere with scaffold stability. The lyophilized 3D-DDS has high rehydration ability and is able to restrain physically bovine bone granules and microparticles up to 45 days in in vitro simulated conditions. Bactericidal action is high in the first 4 h and it accounts for superimposable effect of gentamicin release and bacteriostatic chitosan properties; sustained gentamicin bactericidal effect is evident up to 14 days.

Human Lipoaspirate as Autologous Injectable Active Scaffold for One-Step Repair of Cartilage Defects.

Research on mesenchymal stem cells from adipose tissue shows promising results for cell-based therapy in cartilage lesions. In these studies, cells have been isolated, expanded, and differentiated in vitro before transplantation into the damaged cartilage or onto materials used as scaffolds to deliver cells to the impaired area. The present study employed in vitro assays to investigate the potential of intra-articular injection of micro-fragmented lipoaspirate as a one-step repair strategy; it aimed to determine whether adipose tissue can act as a scaffold for cells naturally present at their anatomical site. Cultured clusters of lipoaspirate showed a spontaneous outgrowth of cells with a mesenchymal phenotype and with multilineage differentiation potential. Transduction of lipoaspirate clusters by lentiviral vectors expressing GFP evidenced the propensity of the outgrown cells to repopulate fragments of damaged cartilage. On the basis of the results, which showed an induction of proliferation and ECM production of human primary chondrocytes, it was hypothesized that lipoaspirate may play a paracrine role. Moreover, the structure of a floating culture of lipoaspirate, treated for 3 weeks with chondrogenic growth factors, changed: tissue with a high fat component was replaced by a tissue with a lower fat component and connective tissue rich in GAG and in collagen type I, increasing the mechanical strength of the tissue. From these promising in vitro results, it may be speculated that an injectable autologous biologically active scaffold (lipoaspirate), employed intra-articularly, may 1) become a fibrous tissue that provides mechanical support for the load on the damaged cartilage; 2) induce host chondrocytes to proliferate and produce ECM; and 3) provide cells at the site of injury, which could regenerate or repair the damaged or missing cartilage.

Evaluation of hydrogel composing of Pluronic F127 and carboxymethyl hexanoyl chitosan as injectable scaffold for tissue engineering applications

This study demonstrated a novel hydrogel system composing of Pluronic F127, carboxymethyl hexanoyl chitosan (CA) and glutaraldehyde (GA) for encapsulating fibroblasts (L-929). The thermal behavior of the hydrogel was evaluated using TGA, the swelling behavior of the hydrogel was evaluated in Dulbecco's Modified Eagle's medium (DMEM), and the mechanical properties were determined through dynamic mechanical analysis. Cells were encapsulated by simple mixing, and the viability of encapsulated cells was determined using alamar blue cell viability assay and the cells morphology was examined using fluorescent imaging. The results indicated that the Tgel of this system was around 30°C, where sol-gel transformation occurred within 90s. Although the addition of CA and GA reduced the shear moduli slightly, the F127/CA/GA gel was able to remain in gelling state in the medium for more than 1 month. In vitro cell culture study revealed that F-127/CA/GA hydrogels were non-cytotoxic. Moreover, the viability of encapsulated L929 was 106% after incubation for 5 days. Based on these results, these F127/CA/GA hydrogels can be used to encapsulate cells for tissue engineering applications.

Novel crosslinkable polyester resin–based composites as injectable bioactive scaffolds

ABSTRACTIn this study new composite materials obtained by crosslinking two polyester resins were investigated. The solid composite material and the scaffolds with varied porosity were obtained in the presence of a foaming system consisted of calcium carbonate/citric acid mixture. It was found that the presence of citric acid solution influenced rheological behavior of liquid formulations as well as morphology and mechanical properties of cured materials. SEM observations and analysis by energy-dispersive X-ray dispersive spectroscopy confirmed the presence of hydroxyapatite formed at the scaffolds surfaces in phosphate buffer solution. The MTS cytotoxicity assay was carried out for extracts from selected composite materials using the human osteosarcoma cells (SaOS-2).

524. Injectable BMP-2 Gene-Activated Scaffold for the Repair of Mouse Cranial Bone Defect

Introduction: A major requirement in bone tissue engineering is the persistent and robust promotion of osteogenesis, which requires sustained stimulation with osteoinductive factors, such as bone morphogenetic protein-2 (BMP-2). However, the direct application of these factors remains challenging due to their short half-life and rapid systemic clearance. Given the well demonstrated benefits of recombinant adeno-associated viral (rAAV) vector, including long-term gene transfer efficiency and relative safety, rAAV-based therapies have been developed in both basic and translational research. However, traditional ex vivo gene transduction process is time consuming and requires multiple steps of cell culture. In addition, rAAV shows inconsistent transfection efficacy in primary cells. In this study, we co-packed rAAV-BMP-2 and human bone marrow stem cells (hBMSCs) into a gelatin scaffolds through one-step visible light-based phtocorsslinking (PXL), and tested their application for bone regenerationi. We hypothesize that the concentrated and localized release of rAAV-BMP-2 from the scaffold matrix results in higher transfection efficacy and BMP-2 expression in hBMSCs seeded within the scaffolds, thus enhancing their osteogenic differentiation and bone formation upon implantation in bone defect. Methods: hBMSCs were obtained with IRB approval from total joint arthroplasty patient. The rAAV6-BMP-2 and rAAV6-GFP reporter (as control) vectors were designed and prepared using an established protocol in our laboratory. PXL was performed using 10% gelatin and 10×106 hBMSCs/ml with different amount of rAAV6-GFP. Transfection efficiency was determined by counting GFP-positive cells ratio as well as assessing cell viability. At an optimal ratio, rAAV-BMP-2 and hBMSCs were included into gelatin through PXL. At different time point, BMP-2 production and rAAV6 release kinetics were analyzed. The osteogenesis of hBMSCs was assessed at day 28 by real time PCR and histological staining. In animal study, a 0.5 cm diameter critical size of cranial bone defect model was generated in SCID mice and then the constructs described above were transplanted. Micro-CT imaging and histological staining were used to evaluate the skull bone healing process up to 6 weeks. Results: Both rAAV and hBMSCs survived during the scaffold fabrication process. 10×104 rAAV/hBMSCs was shown to be the optimal ratio in maintaining cell viability and achieving transduction efficiency. After in situ transfection within scaffolds, hBMSCs produced higher BMP-2 amount than those subjected to traditional ex vivo transduction process, and underwent robust osteogenesis as well. In animal study, micro-CT imaging results indicated that rAAV BMP-2 & hBMSCs-activated gelatin scaffolds effectively promoted bone regeneration in mice cranial bone defect, in a manner significantly superior to that of scaffolds loaded with hBMSCs/BMP-2 protein, and was remarkably compatible to scaffolds loaded with hBMSCs previously ex vivo transduced by rAAV-BMP-2. The histological staining further confirmed the efficacious bone formation from rAAV BMP-2 & hBMSCs loaded scaffold. The results shown here thus demonstrate the feasibility of combining gene and cell therapy with biomaterial engineering via a single-step fabrication and its application for the repair of bone defect.

Modified poly(caprolactone trifumarate) with embedded gelatin microparticles as a functional scaffold for bone tissue engineering

ABSTRACTBone tissue engineering offers high hopes in reconstructing bone defects that result from trauma, infection, tumors, and other conditions. However, there remains a need for novel scaffold materials that can effectively stimulate ossification with appropriate functional properties. Therefore, a novel injectable, biodegradable, and biocompatible scaffold made by incorporating modified poly(caprolactone trifumarate) (PCLTF) with embedded gelatin microparticles (GMPs) as porogen is developed. Specifically, in vitro and in vivo tests were carried out. For the latter, to determine the osteogenic ability of PCLTF‐GMPs scaffolds, and to characterize bone‐formation, these scaffolds were implanted into critical‐sized defects of New Zealand white rabbit craniums. Field Emission Scanning Electron Microscope (FESEM) demonstrated cells of varying shapes attached to the scaffold surface in vitro. The PCLTF‐GMPs demonstrated improved biocompatibility in vivo. Polyfluorochrome tracers detected bone growth occurring in the PCLTF‐GMPs filled defects. By incorporating PCLTF with GMPs, we have fabricated a promising self‐crosslinkable biocompatible and osteoconducive scaffold for bone tissue engineering. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43711.

A thermoresponsive polydiolcitrate-gelatin scaffold and delivery system mediates effective bone formation from BMP9-transduced mesenchymal stem cells

Successful bone tissue engineering requires at the minimum sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin (PPCNG) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line immortalized mouse embryonic fibroblasts (iMEFs), we found that PPCNG facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in a mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs + PPCNG group was significantly more pronounced than that of the direct cell injection group. Accordingly, vascular endothelial growth factor (VEGF) expression was shown to be significantly higher in the bony masses recovered from the iMEFs + PPCNG group. Taken together, our results suggest that PPCNG may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.

Muscle Tissue Engineering Using Gingival Mesenchymal Stem Cells Encapsulated in Alginate Hydrogels Containing Multiple Growth Factors.

Repair and regeneration of muscle tissue following traumatic injuries or muscle diseases often presents a challenging clinical situation. If a significant amount of tissue is lost the native regenerative potential of skeletal muscle will not be able to grow to fill the defect site completely. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material, present an advantageous alternative therapeutic option for muscle tissue engineering in comparison to current treatment modalities available. To date, there has been no report on application of gingival mesenchymal stem cells (GMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of the current study were to develop an injectable 3D RGD-coupled alginate scaffold with multiple growth factor delivery capacity for encapsulating GMSCs, and to evaluate the capacity of encapsulated GMSCs to differentiate into myogenic tissue in vitro and in vivo where encapsulated GMSCs were transplanted subcutaneously into immunocompromised mice. The results demonstrate that after 4weeks of differentiation in vitro, GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited muscle cell-like morphology with high levels of mRNA expression for gene markers related to muscle regeneration (MyoD, Myf5, and MyoG) via qPCR measurement. Our quantitative PCR analyzes revealed that the stiffness of the RGD-coupled alginate regulates the myogenic differentiation of encapsulated GMSCs. Histological and immunohistochemical/fluorescence staining for protein markers specific for myogenic tissue confirmed muscle regeneration in subcutaneous transplantation in our in vivo animal model. GMSCs showed significantly greater capacity for myogenic regeneration in comparison to hBMMSCs (p<0.05). Altogether, our findings confirmed that GMSCs encapsulated in RGD-modified alginate hydrogel with multiple growth factor delivery capacity is a promising candidate for muscle tissue engineering.