Development of injectable high molecular weight hyaluronic acid hydrogels for cartilage regeneration

The study focuses on developing hyaluronic acid (1200 kilo Dalton) hydrogels for cartilage regeneration. In spite of being highly biocompatible; a large amount of water absorption and easily degrading nature restricts the use of hyaluronic acid in the field of tissue regeneration. This can be rectified by crosslinking hyaluronic acid with a crosslinking agent such as divinyl sulfone; which results in a biocompatible hydrogel with superior rheological properties. Different amounts of divinyl sulfone have been used for crosslinking hyaluronic acid to get three types of hydrogels with differing properties. Swelling studies, rheology analysis, enzymatic degradation and scanning electron microscopic analysis were conducted on all the different types of hydrogels prepared. Viscoelastic properties of the hydrogel were analyzed so that a hydrogel with better elastic property and stability is obtained. Scanning electron microscopy was used to study the morphology of the HA hydrogels. The cytotoxicity testing was conducted to prove the non-toxic nature of the hydrogels and cell culture studies using adipose mesenchymal stem cells showed better adhesion and proliferation properties in all the three hydrogels. Thus hyaluronic acid hydrogel makes a promising material for cartilage regeneration.     

Ultrasound augmenting injectable chemotaxis hydrogel for articular cartilage repair in osteoarthritis

The increasing incidence of osteoarthritis (OA) seriously affects life quality, posing a huge socioeconomic burden. Tissue engineering technology has become a hot topic in articular cartilage repair as one of the key treatment methods to alleviate OA. Hydrogel, one of the most commonly used scaffold materials, can provide a good extracellular matrix microenvironment for seed cells such as bone marrow mesenchymal stem cells (BMSCs), which can promote cartilage regeneration. However, the low homing rate of stem cells severely limits their role in promoting articular cartilage regeneration. Stromal cell-derived factor-1α (SDF-1α) plays a crucial role in the activation, mobilization, homing, and migration of MSCs. Herein, a novel injectable chemotaxis hydrogel, composed of chitosan-based injectable hydrogel and embedding SDF-1α-loaded nanodroplets (PFP@NDs-PEG-SDF-1α) was designed and fabricated. The ultrasound was then used to augment the injectable chemotaxis hydrogel and promote the homing migration of BMSCs for OA cartilage repair. The effect of ultrasound augmenting injectable PFP@NDs-PEG-SDF-1α/hydrogel on the migration of BMSCs was verified in vitro and in vivo, which remarkably promotes stem cell homing and the repair of cartilage in the OA model. Therefore, the treatment strategy of ultrasound augmenting injectable chemotaxis hydrogel has a bright potential for OA articular cartilage repair.     

Integration exosomes with MOF-modified multifunctional scaffold for accelerating vascularized bone regeneration

Designing a multifunctional scaffold with osteogenic and angiogenic properties holds promise for ideal bone regeneration. Innovative scaffold was here constructed by immobilizing exosomes derived from human bone mesenchymal stem cells (hBMSCs) onto porous polymer meshes which developed by PLGA and Cu-based MOF (PLGA/CuBDC@Exo). The synthesized exosome-laden scaffold capable of providing a dual cooperative controllable release of bioactive copper ions and exosomes that promote osteogenesis and angiogenesis, thereby achieving cell-free bone regeneration. In vitro assay revealed the composite stent not only substantially upregulated the expression of osteogenic-related proteins (ALP, Runx2, Ocn) and VEGF in hBMSCs, but promoted the migration and tube formation of the human umbilical vein endothelial cells (HUVECs). In vivo evaluation further confirmed this scaffold dramatically stimulated bone regeneration and angiogenesis in critical-sized defects in rats. Altogether, this composite scaffold carrying therapeutic exosomes had an osteogenic-angiogenic coupling effect and offered a new idea for cell-free bone tissue engineering.     

Bioinspired dual dynamic network hydrogels promote cartilage regeneration through regulating BMSC chondrogenic differentiation

How to improve the therapeutic efficacy of cell delivery during mechanical injection has been a great challenge for tissue engineering. Here, we present a facile strategy based on dynamic chemistry to prepare injectable hydrogels for efficient stem cell delivery using hyaluronic acid (HA) and poly(γ-glutamic acid) (γ-PGA). The combination of the guest–host (GH) complexation and dynamic hydrazone bonds enable the HA/γ-PGA hydrogels with physical and chemical dual dynamic network and endow hydrogels a stable structure, rapid self-healing ability, and injectability. The mechanical properties, self-healing ability, and adaptability can be programmed by changing the ratio of GH network to hydrazine bond cross-linked network. Benefitting from the dynamic cross-linking networks, mild preparation process, and cytocompatibility of HA/γ-PGA hydrogels, bone marrow mesenchymal stem cells (BMSCs) show high cell viability in this system following mechanical injection. Moreover, HA/γ-PGA hydrogels can promote BMSC proliferation and upregulate the expression of cartilage-critical genes. Notably, in a rabbit auricular cartilage defect model, BMSC-laden HA/γ-PGA hydrogels can effectively promote cartilage regeneration. Together, we propose a general strategy to develop injectable self-healing HA/γ-PGA hydrogels for effective stem cell delivery in cartilage tissue engineering.     

生物材料表面性能调控骨髓间充质干细胞分化

结合细胞和生物可降解支架的组织工程和再生医学技术为组织、器官的修复和再生提供了一种新途径。骨髓间充质干细胞(BMSCs)具有多向分化潜能,因其取材简单、来源广泛、增殖能力强,无伦理争议,免疫排斥反应小而备受关注。BMSCs在特定区域定向分化成为靶细胞是干细胞治疗的一个重要前提,尤其受到生物材料表面正负电荷、亲疏水和不同的拓扑结构的影响。材料表面涂层蛋白或接枝多肽能够促进BMSCs的分化能力,而生物材料不同的机械性能、几何形状也会影响BMSCs的分化方向。本文综述了近期生物材料调控BMSCs分化的研究结果,为基于BMSCs的组织工程和再生医学材料的设计提供借鉴和指导。     

Dual Delivery of TGF-β3 and Ghrelin in Microsphere/Hydrogel Systems for Cartilage Regeneration

Articular cartilage (AC) damage is quite common, but due to AC’s poor self-healing ability, the damage can easily develop into osteoarthritis (OA). To solve this problem, we developed a microsphere/hydrogel system that provides two growth factors that promote cartilage repair: transforming growth factor-β3 (TGF-β3) to enhance cartilage tissue formation and ghrelin synergy TGF-β to significantly enhance the chondrogenic differentiation. The hydrogel and microspheres were characterized in vitro, and the biocompatibility of the system was verified. Double emulsion solvent extraction technology (w/o/w) is used to encapsulate TGF-β3 and ghrelin into microspheres, and these microspheres are encapsulated in a hydrogel to continuously release TGF-β3 and ghrelin. According to the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) in vitro, the concentrations of the two growth factors were optimized to promote cartilage regeneration.     

Boswellic acid captivated hydroxyapatite carboxymethyl cellulose composites for the enhancement of chondrocytes in cartilage repair

Recently tissue repairing bone grafted materials have been greater properties than the recapitulating intramembranous regeneration of natural bone especially cartilage damage regeneration. In this present work was designed and developed for the enhancement of chondrocyte generation for cartilage repair. Boswellic acid (BA) is a traditional compound used for the treatment of osteoarthritis. Boswellic acid subjected to involve the preparation of hydroxyapatite (HAP) and HAP-BA compounds were functionalized with carboxymethyl cellulose (CMC) to promote the extra-cellular matrix. HAP, HAP-BA and HAP-BA-CMC composites were characterized via their physicochemical properties through FTIR, XRD, SEM and TEM techniques. The antibacterial activity and chondrocyte cell variability were tested. At 14 days, HAP-BA-CMC composite was observed 67% of cell viability. The chondrocyte cell adhesion on the HAP-BA-CMC composite was investigated and it observed polygonal filopodia. From the results suggest that HAP-BA-CMC composite can improve the chondrocyte production for repair of damaged cartilage.     

Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder

The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application.     

Enhanced Bone Defect Repair by Polymeric Substitute Fillers of MultiArm Polyethylene Glycol‐Crosslinked Hyaluronic Acid Hydrogels

Bone regeneration is still one of the greatest challenges for the treatment of bone defects since no current clinical approach has been proven effective. To develop an alternative biodegradable bone graft material, multiarm polyethylene glycol (PEG) crosslinked hyaluronic acid (HA) hydrogels are synthesized and applied to promote osteogenesis of mesenchymal stem cells (MSCs) with the ultimate goal for bone defect repair. The multiarm PEG‐HA hydrogels provide a significant improvement of alkaline phosphatase (ALP) activity and calcium mineralization of the in vitro encapsulated MSCs under osteogenic condition after 3, 7, and 28 days. In addition, the multiarm PEG‐HA hydrogels also facilitate healing of the cranial bone defects more effectively in a Sprague Dawley rat model after 10 weeks of implantation based on histological evaluations and microcomputed tomography analysis. These promising results set the stage for the development of innovative biodegradable hydrogels to provide a more effective and versatile treatment option for bone regeneration.     

Homogeneous organic/inorganic hybrid scaffolds with high osteoinductive activity for bone tissue engineering

Composite scaffolds of polymers/β-tricalcium phosphate (TCP) have been widely used for bone regeneration due to the combination of osteoinductivity of TCP and mechanical properties of the polymers. However, the difference in surface properties of the two material causes composite has poor uniformity and weak two-phase interaction, resulting in poor TCP release and weak new bone-forming ability. In this research, a TCP sol was developed to replace traditional TCP nanoparticles for the preparation of homogeneous polycaprolactone (PCL)/TCP sol nanofibrous scaffolds. It was found that compared with TCP nanoparticles, TCP sol homogeneously distributed in PCL nanofibers, and greatly improved the hydrophilicity, biodegradability, and mechanical properties of the scaffolds. It is also confirmed that loading TCP sol promoted the formation of bone-like apatite on the surface of the scaffolds. Biological experiments showed that all scaffolds supported rat bone marrow mesenchymal stem cells (rBMSCs) proliferation, especially scaffolds loaded with TCP sol. The increase in alkaline phosphatase activity and collagen production, enhanced calcium deposition, and up-regulation of osteocalcin expression demonstrated that the loading TCP sol expanded an advantage of scaffolds in promoting rBMSCs osteogenic differentiation, suggesting it dramatically improved the osteoinductive activity of PCL/TCP hybrid system and had a great potential application in bone regeneration.     

Effect of F68 on Cryopreservation of Mesenchymal Stem Cells Derived from Human Tooth Germ

The use of stem-cell-based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimer's disease, diabetes, spinal cord injuries, and cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory, and angiogenic effects which are attributed to the restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Unlike bone-marrow-derived MSCs, HTGSCs can be easily isolated, expanded, and cryopreserved, which makes them an alternative stem cell source. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation, hindering their therapeutic capacity. Amelioration of the side effects of cryopreservation on MSCs seems to be a priority in order to maximize the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F68) on HTGSCs during long-term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long-term cryopreservation.     

Recruitment of endogenous stem cells for tissue repair

The traditional concept of stem cell therapy envisions the isolation of stem cells from patients, propagation and differentiation in vitro, and subsequent re-injection of autologous cells into the patient. There are many problems associated with this paradigm, particularly during the in vitro manipulation process and the delivery and local retention of re-injected cells. An alternative paradigm that could be easier, safer, and more efficient, would involve attracting endogenous stem cells and precursor cells to the defect site for new tissue regeneration. Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin, exerts a strong chemoattractive effect on mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and induces migration of MSCs in vitro. However, HGF undergoes rapid proteolysis in vivo, which results in a very short lifetime of the bioactive cytokine. To maintain the therapeutic level of HGF at the defect site necessary for endogenous stem cell recruitment, sustained, long-term, and localized delivery of HGF is required. Thiol-modified glycosaminoglycans hyaluronan (HA) and heparin (HP), combined with modified gelatin (Gtn), have been crosslinked with poly(ethylene glycol) diacrylate (PEGDA) to afford semisynthetic ECM-like (sECM) hydrogels that can both provide controlled growth factor release and permit cell infiltration and proliferation. Herein we compare the use of different sECM compositions for controlled release of HGF and concomitant recruitment of human bone marrow MSCs into the scaffold in vitro. [Figure: see text].     

Preparation and characterization of composite nanofibers of polycaprolactone and nanohydroxyapatite for osteogenic differentiation of mesenchymal stem cells

Nanocomposites of nanohydroxyapatite (nHAP) dispersed in poly(?-caprolactone) (PCL) were prepared by electrospinning (ES) to obtain PCL/nHAP nanofibers. Nanofibers with similar diameters (340 ± 30 nm) but different nHAP concentrations (0-50%) were fabricated and studied for growth and osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). The nanofibrous membranes were subjected to detailed analysis for its physicochemical properties by scanning electron microscopy (SEM), thermogravimetric analysis, X-ray diffraction, Fourier-transform infrared spectroscopy, and mechanical tensile testing. nHAP particles (~30 nm diameter) embedded in nanofibers increased the nanofibrous membrane's ultimate stress and the elastic modulus, while decreased the strain at failure. When cultured under an osteogenic stimulation condition on nanofibers, MSCs showed normal phenotypic cell morphology, and time-dependent mineralization and osteogenic differentiation from SEM observations and alkaline phosphatase activity assays. The nanofibers could support the growth of mesenchymal stem cells without compromising their osteogenic differentiation capability up to 21 days and the enhancement of cell differentiation by nHAP is positively correlated with its concentration in the nanofibers. Energy dispersive X-ray analysis of Ca and P elements indicated mineral deposits on the cell surface. The mineralization extent was significantly raised in nanofibers with 50% nHAP where a Ca/P ratio similar to that of bone was found. The present study indicated that electrospun composite PCL/nHAP nanofibrous membranes are suitable for mineralization of MSCs intended for bone tissue engineering.     

Controlling the Poly(ε‐caprolactone) Degradation to Maintain the Stemness and Function of Adipose‐Derived Mesenchymal Stem Cells in Vascular Regeneration Application

Biodegradable poly(ε‐caprolactone) (PCL) scaffolds with adipose‐derived mesenchymal stem cells (ADSCs) have been used in vascular regeneration studies. An evaluation method of the effect of PCL degradation products (DP) on the viability, stemness, and differentiation capacities of ADSCs is established. ADSCs are cultured in medium containing different concentrations of PCL DP before evaluating the effect of PCL DP on the cell apoptosis and proliferation, cell surface antigens, adipogenic and osteogenic differentiation capacities, and capacities to differentiate into endothelial cells and smooth muscle cells. The results demonstrate that PCL DP exceed 0.05 mg mL^?1 may change the stemness and differentiation capacities of ADSCs. Therefore, to control the proper concentration of PCL DP is essential for ADSCs in vascular regeneration application.     

Laponite–Gelatin Nanofibrous Microsphere Promoting Human Dental Follicle Stem Cells Attachment and Osteogenic Differentiation for Noninvasive Stem Cell Transplantation

Nanofibrous microspheres (NFM) are emerging as prominent next-generation biomimetic injectable scaffold system for stem cell delivery and different tissue regeneration where nanofibrous topography facilitates ECM-like stem cells niches. Addition of osteogenic bioactive nanosilicate platelets within NFM can provide osteoconductive cues to facilitate matrix mediated osteogenic differentiation of stem cells and enhance the efficiency of bone tissue regeneration. In this study, gelatin nanofibrous microspheres are prepared containing fluoride-doped laponite XL21 (LP) using the emulsion mediated thermal induce phase separation (TIPS) technique. Systematic studies are performed to understand the effect of physicochemical properties of biomimicking NFM alone and with different concentrations of LP on human dental follicle stem cells (hDFSCs), their cellular attachment, proliferation, and osteogenic differentiation. The study highlights the effect of LP nanosilicate with biomimicking nanofibrous injectable scaffold system aiding in enhancing stem cell differentiation under normal physiological conditions compared to NFM without LP. The laponite–NFM shows suitability as excellent injectable biomaterials system for stem cell attachment, proliferation and osteogenic differentiation for stem cell transplantation and bone tissue regeneration.     

Progress in Articular Cartilage Tissue Engineering: A Review on Therapeutic Cells and Macromolecular Scaffolds

Repair and regeneration of articular cartilage lesions have always been a major challenge in the medical field due to its peculiar structure (e.g., sparsely distributed chondrocytes, no blood supply, no nerves). Articular cartilage tissue engineering is considered as one promising strategy to achieve reconstruction of cartilage. With this perspective, the articular cartilage tissue engineering has been widely studied. Here, the recent progress of articular cartilage tissue engineering is reviewed. The ad hoc therapeutic cells and growth factors for cartilage regeneration are summarized and discussed. Various types of bio/macromolecular scaffolds together with their pros and cons are also reviewed and elaborated.     

A fast on-demand preparation of injectable self-healing nanocomposite hydrogels for efficient osteoinduction

Injectable hydrogels have been considered as promising materials for bone regeneration,but their osteoinduction and mechanical performance are yet to be improved.In this study,a novel biocompatible injectable and self-healing nano hybrid hydrogel was on-demand prepared via a fast(within 30 s) and easy gelation approach by reversible Schiff base formed between-CH=O of oxidized sodium alginate(OSA) and-NH_2 of glycol chitosan(GCS) mixed with calcium phosphate nanoparticles(CaP NPs).Its raw materials can be ready in large quantities by a simple synthesis process.The mechanical strength,degradation and swelling behavior of the hydrogel can be readily controlled by simply controlling the molar ratio of-CH=O and-NH_2.This hydrogel exhibits pH responsiveness,good degradability and biocompatibility.The hydrogel used as the matrix for mesenchymal stem cells can significantly induce the proliferation,differentiation and osteoinduction in vitro.These results showed this novel hydrogel is an ideal candidate for applications in bone tissue regeneration and drug delivery.     

Intramarrow injection of β-catenin-activated,but not na?ve mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naïve MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with β-catenin-activated MSCs. These results reveal that naïve MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/β-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.