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Large bone defects that cannot form a callus tissue are often faced with long-time recovery. Developmental engineering-based strategies with mesenchymal stem cell (MSC) aggregates have shown enhanced potential for bone regeneration. However, MSC aggregates are different from the physiological callus tissues, which limited the further endogenous osteogenesis. This study aims to achieve engineering of osteo-callus organoids for rapid bone regeneration in cooperation with bone marrow-derived stem cell (BMSC)-loaded hydrogel microspheres (MSs) by digital light-processing (DLP) printing technology and stepwise-induction. The printed MSC-loaded MSs aggregated into osteo-callus organoids after chondrogenic induction and showed much higher chondrogenic efficiency than that of traditional MSC pellets. Moreover, the osteo-callus organoids exhibited stage-specific gene expression pattern that recapitulated endochondral ossification process, as well as a synchronized state of cell proliferation and differentiation, which highly resembled the diverse cell compositions and behaviors of developmentally endochondral ossification. Lastly, the osteo-callus organoids efficiently led to rapid bone regeneration within only 4 weeks in a large bone defect in rabbits which need 2-3 months in previous tissue engineering studies. The findings suggested that in vitro engineering of osteo-callus organoids with developmentally osteogenic properties is a promising strategy for rapid bone defect regeneration and recovery.

Although biomaterials are widely utilized in clinics, it still follows the "one-fits-all" strategy. Biological variables such as age and sexuality have an impact on the host immune response and are not fully considered in the practice guidelines of the biomaterial implantation. In this study, we investigated the immuno-material interactions of six commonly used biomaterials (agarose, alginate, chitosan, CMC, GelMA and collagen type I) and constructed a population (with different ages and sexes) based transcriptome atlas. Protein and polysaccharide-based biomaterials elicited distinctive immune responses that protein-based materials preferred the NKT pathway to activate innate and adaptive immune response, whereas polysaccharide-based materials activated the cDCs to present antigen. The atlas further revealed the sex/age-related variabilities on the immune response followed by the polysaccharide treatment. As for sex bias, alginate and agarose stimulation significantly increased the proportion of naive CD4+ T cells in the female group, accompanied by the Th1 differentiation tendency, compared to the male group. Age-biased transcript showed alginate and chitosan would impair the extracellular matrix remodeling and up-regulate the apoptosis process in the elderly groups, compared to the young group. More attentions on the ingredient, age and sexuality effect of biomaterial implants should be paid during the clinical practice, especially for the polysaccharide-based materials. This experimental result is of great significance for the selection of biomaterials, particularly the blood contact materials, such as vessel or cardiac device, drug vehicles and hemostatic materials.

Macrophages play crucial roles in host tissue reaction to biomaterials upon implantation in vivo. However, the complexity of biomaterial degradation-related macrophage subpopulations that accumulate around the implanted biomaterials in situ is not fully understood. Here, using single cell RNA-seq, we analyze the transcriptome profiles of the various cell types around the scaffold to map the scaffold-induced reaction, in an unbiased approach. This enables mapping of all biomaterial degradation-associated cells at high resolution, revealing distinct subpopulations of tissue-resident macrophages as the major cellular sources of biomaterial degradation in situ. We also find that scaffold architecture can affect the mechanotransduction and catabolic activity of specific material degradation-related macrophage subpopulations in an Itgav-Mapk1-Stat3 dependent manner, eventually leading to differences in scaffold degradation rate in vivo. Our work dissects unanticipated aspects of the cellular and molecular basis of biomaterial degradation at the single-cell level, and provides a conceptual framework for developing functional tissue engineering scaffolds in future.

Biomaterials are indispensable for tissue engineering, which plays a pivotal role in the skeletal tissue repair. However, biomaterials currently used such as animal extracts and chemically synthesized polymers display unsatisfactory bioactivity and safety. In recent years, modular protein engineering-based (MPE) biomaterials composed of polypeptides produced by molecular cloning and protein synthesis have greatly developed due to their lower batch-to-batch variation, avoidance of possible pathogens and, most importantly, sequence-tunable property. In this review, we first briefly describe the properties of different MPE biomaterials classified by the structural domains of polypeptides, and techniques to engineer the polypeptide sequence and synthesize MPE biomaterials at will. Then, we focus on the application of bio-designed MPE biomaterials in skeletal tissue engineering. Different structural domains of polypeptides are used individually or covalently fused with different bioactive motifs to generate a variety of MPE biomaterials. The sequence (protein modules) of MPE biomaterials would determine and guide their cytocompatibility, their effects on cell fate and ECM formation, the mechanical properties and functions during the in vivo skeletal tissue repair. Moreover, we propose several bio-design strategies and potential directions to develop MPE biomaterials for better performing skeletal tissue engineering and to achieve fast skeletal tissue regeneration. Combinations of material science and protein engineering would provide solutions to the obstacles in regenerative medicine. This article provides a board review of skeletal tissue engineering in a polypeptide sequence-guided way by using MPE biomaterials.

Great effort has been spent to promote the vascularization of tissue engineering bone grafts (TEBG) for improved therapeutic outcome. However, the thorough vascularization especially in the central region still remained as a major challenge for the clinical translation of TEBG. Here, we developed a new strategy to construct a centrally vascularized TEBG (CV-TEBG) with unique core-shell composite structure, which is consisted of an angiogenic core and an osteogenic shell. The in vivo evaluation in rabbit critical sized femoral defect was conducted to meticulously compare CV-TEBG to other TEBG designs (TEBG with osteogenic shell alone, or angiogenic core alone or angiogenic core+shell). Microfil-enhanced micro-CT analysis has been shown that CV-TEBG could outperform TEBG with pure osteogenic or angiogenic component for neo-vascularization. CV-TEBG achieved a much higher and more homogenous vascularization throughout the whole scaffold (1.52-38.91 folds, p < 0.01), and generated a unique burrito-like vascular network structure to perfuse both the central and peripheral regions of TEBG, indicating a potential synergistic effect between the osteogenic shell and angiogenic core in CV-TEBG to enhance neo-vascularization. Moreover, CV-TEBG has generated more new bone tissue than other groups (1.99-83.50 folds, p < 0.01), achieved successful bridging defect with the formation of both cortical bone like tissue externally and cancellous bone like tissue internally, and restored approximately 80% of the stiffness of the defected femur (benchmarked to the intact femur). It has been further observed that different bone regeneration patterns occurred in different TEBG implants and closely related to their vascularization patterns, revealing the potential profound influence of vascularization patterns on the osteogenesis pattern during defect healing.

Three dimensional (3D) microenvironments more accurately replicate native microenvironments for stem cell maintenance and function compared with two dimensional (2D) microenvironments. However, the molecular mechanisms by which 3D microenvironments regulate stem cell function remain largely unexplored at the single-cell level. Here, using a single-cell analysis and functional analysis, we found not all cell-subpopulations respond to 3D microenvironments based on a systematically 3D gelatin microcarrier culture system we developed for the expansion and function maintenance of hTSPCs. 3D microenvironments alter the cell-subpopulation distribution of human tendon stem/progenitor cells (hTSPCs) by improving the proportion of ICAM1+ITGB8+ and FGF7+CYGB+ subpopulations. We also revealed the activated FGF7 signaling in the two subpopulations is responsible for the enhanced tenogenesis of hTSPCs through cell-cell interactions. The hTSPCs cultured in 3D niche with a specific cell-subpopulation structure exhibited superior stem-cell characteristics and functions both in vitro and in vivo. Together, our study demonstrates that 3D microenvironments can regulate stem-cell function by modulating the critical cell subpopulation and identifies FGF7 as a novel regulator for tenogenic differentiation and tendon regeneration.

It is still a challenge for existing bioprinting technologies to fabricate organs suitable for implantation, mainly due to the inability to recapitulate the organs' complex anatomical structures, mechanical properties, and biological functions. Additionally, the failure to create 3D constructs with interconnected microchannels for long-range mass transportation that limits the clinical applications of 3D printing technologies. Here, a new method was developed to print functional living skin (FLS) using a newly designed biomimetic bioink (GelMA/HA-NB/LAP) and digital light processing (DLP)-based 3D printing technology. The FLS possess interconnected microchannels that facilitates cell migration, proliferation and neo-tissue formation. The GelMA/HA-NB/LAP bioink, composed of gelatin methacrylate (GelMA), N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide (NB) linked hyaluronic acid (HA-NB) and photo-initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The bioink demonstrated its rapid gelation kinetics, tunable mechanical properties, good biocompatibility and tissue adhesion. The DLP-based 3D printing technology provides a rapid method to precisely position clusters of human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs) with high cell viability to form FLS. The FLS promotes skin regeneration and efficient neovascularization by mimicking the physiological structure of natural skin, and it can also be easily handled and implanted onto the wound site due to its strong mechanical and bio-adhesive properties. Moreover, in vivo study demonstrated that the living skin exhibited instant defense function and had superior performance in promoting dermal regeneration with skin appendages in large animals. This study provides a rapid and mass production method of functional living organs for future clinical applications.

Osteoarthritis (OA) is a major cause of disability and socioeconomic loss worldwide. However, the current pharmacological approaches used to treat OA are largely palliative. Being the hallmark of OA, the cartilage extracellular matrix (ECM) destruction and abnormal homeostasis is gaining more attention as a therapeutic target in cartilage regeneration. Moreover, during the progression of OA, the cartilage ECM shows significant pathological alternations, which can be promising biomarkers in identifying the pathological stages of OA. In this review, we summarize the role of abnormal ECM homeostasis in the joint cartilage during OA. Furthermore, we provide an update on the cartilage ECM derived biomarkers and regenerative medicine therapies targeting cartilage ECM which includes preclinical animal models study and clinical trials.

Changes in the stiffness of chondrocyte extracellular matrix (ECM) are involved in the pathological progression of osteoarthritis (OA). However, the downstream responses of cartilage ECM stiffness are still unclear. YAP (Yes-associated protein) has been extensively studied as a mechanotransducer, we thus hypothesized that by targeting the downstream molecule activity of ECM stiffness could maintain chondrocyte phenotype and prevent cartilage degeneration in OA. Here, we showed that human cartilage matrix stiffened during pathological progression of OA, and the chondrocyte YAP activity was associated with ECM stiffness. We then mimicked the physiological and pathological stiffness of human cartilage by using PDMS-based substrates, and found that YAP was activated in chondrocytes seeded on stiff substrate, gradually losing their phenotype. In addition, it was observed that YAP was also significantly activated in mice OA development, and conditional knockout (cKO) of YAP in mice preserved collagen II expression and protected cartilage from degeneration in the OA model. Furthermore, intra-articular injection of YAP-selective inhibitor, Verteporfin, significantly maintained cartilage homeostasis in mice OA model. This study indicates that the application of mechanotransducer-targeted drugs could be a potential therapeutic approach for cartilage repair in OA.

Nanoparticles are widely developed and utilized in the pharmaceutical and medicine industry, as they can be easily distributed and infiltrated throughout the whole body once administered; however, the body wide effect of nanoparticles infiltration is still unclear. In this study, we developed a new strategy of Nano Genome Altas (NGA) of multi-tissues to study the acute Body-wide-Organ-Transcriptomic response to nanomaterials. Hydroxyapatite(HA)-Nanoparticles (HANPs) was applied in this study as an example both in vitro and in vivo. Results showed that the effect of HANPs is organ specific and mainly related to immune responses in spleen and muscle, proliferation in spleen and bone, stress and apoptosis in spleen and PBMC, ion transport in spleen, kidney, and liver tissues, metabolism in heart, spleen, and muscle, as well as tissue specific epigenetic and signal pathways. In vitro experiments also confirmed that the effects of HANPs on different tissue stem cells were tissue specific. Thus, Nano Genome Altas can provide a body-wide view of the transcriptomic response of multiple organs and tissue specific stem cells to HANPs; it could also be useful for optimizing HANPs and other nano-delivery systems.

Osteochondral defects are most commonly characterized by damages to both cartilage and bone tissues as a result of serious traumas or physical diseases; because these two tissues have their own unique biological properties, developing a single monophasic scaffold that can concurrently regenerate these two specific lineages becomes a challenge. To address this concern, a silicon-based bioceramic (SiCP) scaffold was fabricated. The efficiency and underlying mechanisms of SiCP for osteochondral defect regeneration were investigated. At 8 and 16 weeks post-implantation in a rabbit model of osteochondral defect, gross morphology, histological, and micro-CT images showed that SiCP scaffolds distinctly promoted subchondral bone and cartilage regeneration when compared to calcium-phosphate based bioceramics (CP) scaffolds without silicon. In vitro, SiCP was also shown to promote bone marrow stem cells (BMSC) osteogenesis (ALP, RUNX2, OCN) and help maintain chondrocytes phenotype (Acan, Sox9, Col2a1), validated by qPCR, western blot, and RNA-sequencing (RNA-seq). Additionally, the descriptive analysis of RNA-seq using Gene Ontology (GO) and KEGG pathway analysis revealed biological processes related to cartilage and bone development and extracellular matrices in chondrocytes, as well as related to early osteogenesis in BMSC, indicating that Si ions play an important role in the regeneration of both tissues. Conclusively, the development of silicon-based bioceramic scaffolds may be a promising approach for osteochondral defect regeneration due to their unique dual-lineage bioactivity.

Physical property of substrates such as stiffness and topography have been reported to induce mesenchymal stem cells differentiation into bone, muscle and neuron lineages. Human-induced pluripotent stem cells (hiPSCs) are a highly promising cell source for regenerative medicine. However, physical properties have not yet been reported to successfully induce pluripotent stem cells into specific lineages. This study aimed to develop a robust, stepwise topographic strategy to induce hiPSCs differentiate into teno-lineage. A novel spinning approach termed stable jet electrospinning (SJES), is utilized to fabricate continuous well-aligned ultrafine fibers (891 ± 71 nm), which mimic the native tendon's microstructure and mechanical properties. hiPSCs are first differentiated into MSCs on smooth plastic surface as confirmed by the differentiations into three mesenchymal lineages and expression of characteristic MSC surface markers through an EMT (Epithelial–Mesenchymal Transition) process. Subsequently, the hiPSC derived MSCs are seeded onto well-aligned fibers to differentiate into tenocyte-like cells through activating mechanic-signal pathway. The in situ tendon repair study further confirms that aligned fiber scaffold with hiPSC-MSCs had significant effect on improving the structural and mechanical properties of tendon injury repair. These findings indicate that the stepwise physical substrate change strategy can be adopted to induce hiPSCs differentiation for tendon tissue regeneration.

Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.

The migration of cells from the side and the bottom of the defect is important in osteochondral defect healing. Here, we designed a novel collagen scaffold that possessed channels in both the horizontal and the vertical directions, along with stromal cell-derived factor-1 (SDF-1) to enhance osteochondral regeneration by facilitating cell homing. Firstly we fabricated the radially oriented and random collagen scaffolds, then tested their properties. The radially oriented collagen scaffold had better mechanical properties than the random scaffold, but both supported cell proliferation well. Then we measured the migration of BMSCs in the scaffolds in vitro. The radially oriented collagen scaffold effectively promoted their migration, and this effect was further facilitated by addition of SDF-1. Moreover, we created osteochondral defects in rabbits, and implanted them with random or oriented collagen scaffolds with or without SDF-1, and evaluated cartilage and subchondral bone regeneration at 6 and 12 weeks after surgery. Cartilage regeneration with both the radially oriented scaffold and SDF-1 effectively promoted repair of the cartilage defect. Our results confirmed that the implantation of the radially oriented channel collagen scaffold with SDF-1 could be a promising strategy for osteochondral repair.

Tendon stem/progenitor cells (TSPCs) have been identified as a rare population in tendons. In vitro propagation is indispensable to obtain sufficient quantities of TSPCs for therapies. However, culture-expanded TSPCs are prone to lose their phenotype , resulting in an inferior repaired capability. And little is known about the underlying mechanism. Here, we found that altered gene expression was associated with increased histone deacetylase (HDAC) activity and expression of HDAC subtypes. Therefore, we exposed ScxGFP mice-derived TSPCs to HDAC inhibitor (HDACi) trichostatin A (TSA) or valproic acid (VPA), and observed significant expansion of ScxGFP+ cells without altering phenotypic properties. TSA upregulated Scx expression by inhibiting HDAC1 and -3, and increasing the H3K27Ac level of Tgfb1 and -2 genome region. Additionally, cell sheets formed from TSA-pretreated mTSPCs retained the ability to accelerate tendon repair in vivo. Thus, our results uncovered an unrecognized role of HDACi in phenotypic and functional mTSPCs expansion to enhance their therapeutic potential.

Intervertebral disc (IVD) degeneration is one of the most widespread musculoskeletal diseases worldwide, which remains an intractable clinical challenge. The aim of this study is to investigate the therapeutic potential of the small molecule gefitinib (an epidermal growth factor receptor (EGFR) inhibitor) in ameliorating IVD degeneration. Aberrant EGFR activation levels were detected in both human and rat degenerative IVDs, which prompted us to investigate the functional roles of EGFR by utilizing inducible cartilage-specific EGFR-deficient mice. We demonstrated that conditional EGFR deletion in mice increased nucleus pulposus (NP) extracellular matrix (ECM) production and autophagy marker activation while MMP13 expression decreased. These outcomes are comparable to the use of a controlled-release injectable thermosensitive hydrogel of gefitinib to block EGFR activity in a puncture-induced rat model. We also conducted a case series study involving patients with non-small cell lung cancer and IVD degeneration who received gefitinib treatment from 2010 to 2015. Gefitinib-treated patients displayed a relative slower disc degenerating progression, in contrast to control subjects. These findings thus provide evidence that suppression of EGFR by the FDA-approved drug gefitinib can protect IVD degeneration in rats, implying the potential application of gefitinib as a small molecule drug for treating IVD degeneration.

In this study we developed a tissue engineered bulking agent that consisted of adipose-derived stem cells (ADSCs) and silk fibroin microspheres to treat stress urinary incontinence caused by severe intrinsic sphincter deficiency (ISD). ISD models were established by completely transection of the bilateral pudendal nerve (PNT) and confirmed by the decreased leak-point pressure (LPP) and increased lumen area of urethra. Injection of silk fibroin microspheres could recover LPP and lumen area at 4 weeks but its efficacy disappears at 8, 12 weeks. Moreover, it was exciting to find that tissue engineered bulking agent brought long-term efficacy (at 4, 8, 12 weeks post-injection) on the recovery of LPP and lumen area. Concomitantly with the function, tissue engineered bulking agent treated group also improved the urethral sphincter structure as exhibited by better tissue regeneration. The findings showed that silk fibroin microspheres alone could work effectively in short-term, while tissue engineered bulking agent that combined silk fibroin microspheres with ADSCs exhibited promising long-term efficacy. This study developed a new strategy of tissue engineered bulking agent for future ISD therapy.

The repair of osteochondral defects can be enhanced with scaffolds but is often accompanied with undesirable terminal differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Parathyroid hormone-related protein (PTHrP) has been shown to inhibit aberrant differentiation, but administration at inappropriate time points would have adverse effects on chondrogenesis. This study aims to develop an effective tissue engineering strategy by combining PTHrP and collagen-silk scaffold for osteochondral defect repair. The underlying mechanisms of the synergistic effect of combining PTHrP administration with collagen-silk scaffold implantation for rabbit knee joint osteochondral defect repair were investigated. In vitro studies showed that PTHrP treatment significantly reduced Alizarin Red staining and expression of terminal differentiation-related markers. This is achieved in part through blocking activation of the canonical Wnt/β-catenin signaling pathway. For the in vivo repair study, intra-articular injection of PTHrP was carried out at three different time windows (4-6, 7-9 and 10-12 weeks) together with implantation of a bi-layer collagen-silk scaffold. Defects treated with PTHrP at the 4-6 weeks time window exhibited better regeneration (reconstitution of cartilage and subchondral bone) with minimal terminal differentiation (hypertrophy, ossification and matrix degradation), as well as enhanced chondrogenesis (cell shape, Col2 and GAG accumulation) compared with treatment at other time windows. Furthermore, the timing of PTHrP administration also influenced PTHrP receptor expression, thus affecting the treatment outcome. Our results demonstrated that intra-articular injection of PTHrP at 4-6 weeks post-injury together with collagen-silk scaffold implantation is an effective strategy for inhibiting terminal differentiation and enhancing chondrogenesis, thus improving cartilage repair and regeneration in a rabbit model.

In bone tissue engineering, a combination of biomimetic nanofibrous scaffolds with renewable stem cells has recently emerged as a new strategy for promoting bone regeneration. We have previously developed a biomimetic nanocomposite nanofibrous scaffold of hydroxyapatite/chitosan (nHAp/CTS) [1]. However, the mechanism behind the supportive function of the scaffolds has not yet been adequately explored. Here, we evaluated the effect of nHAp/CTS seeded with bone marrow mesenchymal stem cells (BMSCs) on bone regeneration and examined the underlying mechanism in vitro and in vivo. The scaffolds of nHAp/CTS induced higher proliferation of BMSCs than membranous hydroxyapatite/chitosan (mHAp/CTS) and electrospun nanofibrous chitosan (nCTS) did. Interestingly, regardless the nanfibrous effect, nHAp/CTS and mHAp/CTS supported the spindle-shaped morphology, in contrast to the spherical shape of BMSCs on nCTS, indicating that HAp supports cell adhesion. Furthermore, the levels of the mRNA for Smad1, BMP-2/4, Runx2, ALP, collagen I, integrin subunits together with myosins were significantly up-regulated on nHAp/CTS whereas these genes were expressed at markedly low levels on mHAp/CTS and nCTS even in osteogenic medium. In addition, the critical proteins pSmad1/5/8 in BMP pathway showed clear nuclear localization and osteocalcin were significantly elevated on nHAp/CTS than mHAp/CTS (P < 0.01) and nCTS (P < 0.01). Similarly, the cells exhibited higher ALP activity on nHAp/CTS than mHAp/CTS (P < 0.01) and nCTS (P < 0.05). Therefore, the findings indicated the activating of intergrin-BMP/Smad signaling pathway of BMSCs on nHAp/CTS. Finally, in vivo, nHAp/CTS/BMSCs had a superior ability of bone reconstruction than other groups for cranial bone defects. In conclusion, our results demonstrated that nHAp/CTS scaffold promotes bone regeneration by supporting the adhesion, proliferation and activating integrin-BMP/Smad signaling pathway of BMSCs both in vitro and in vivo.

Despite the presence of cartilage-derived mesenchymal stem cells (C-MSCs) and synovial membrane-derived mesenchymal stem cells (SM-MSCs) populations, partial-thickness cartilage defects, in contrast to the full-thickness defects, are devoid of spontaneous repair capacity. This study aims to create an in situ matrix environment conducive to C-MSCs and SM-MSCs to promote cartilage self-repair. Spontaneous repair with MSCs migration into the defect area was observed in full-thickness defects, but not in partial-thickness defects in rabbit model. Ex vivo and in vitro studies showed that subchondral bone or type 1 collagen (col1) scaffold was more permissive for MSCs adhesion than cartilage or type 2 collagen (col2) scaffold and induced robust stromal cell-derived factors-1 (SDF-1) dependent migration of MSCs. Furthermore, creating a matrix environment with col1 scaffold containing SDF-1 enhanced in situ self-repair of partial-thickness defects in rabbit 6 weeks post-injury. Hence, the inferior self-repair capacity in partial-thickness defects is partially owing to the non-permissive matrix environment. Creating an in situ matrix environment conducive to C-MSCs and SM-MSCs migration and adhesion with col1 scaffold containing SDF-1 can be exploited to improve self-repair capacity of cartilage.

Poly(lactide-co-glycolide) (PLGA) sponge was filled with fibrin gel, bone marrow mesenchymal stem cells (BMSCs) and transforming growth factor-β1 (TGF-β1) to obtain a construct for cartilage restoration in vivo. The PLGA sponge lost its weight steadily in vitro, but degraded much faster in the construct of PLGA/fibrin gel/BMSCs implanted in the full-thickness cartilage defects. The in vivo degradation of the fibrin gel inside the construct was prolonged to 12 wk too. The CM-DiI labeled allogenic BMSCs were detectable after transplantation (implantation) into the defects for 12 wk by small animal in vivo fluorescence imaging and confocal laser scanning microscopy. In vivo repair experiments were firstly performed by implantation of the PLGA/fibrin gel/BMSCs and PLGA/BMSCs constructs into full-thickness cartilage defects (3 mm in diameter and 4 mm in depth) of New Zealand white rabbits for 12 wk. The defects implanted with the PLGA/fibrin gel/BMSCs constructs were filled with cartilage-like tissue containing collagen type II and glycosaminoglycans (GAGs), while those by the PLGA/BMSCs constructs were filled with fibrous-like tissues. To repair the defects of larger size (4 mm in diameter), addition of growth factors was mandatory as exemplified here by further loading of TGF-β1. Implantation of the PLGA/fibrin gel/BMSCs/TGF-β1 constructs into the full-thickness cartilage defects for 12 wk resulted in full restoration of the osteochondral tissue. The neo-cartilage integrated well with its surrounding cartilage and subchondral bone. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the cartilage special genes were significantly up-regulated compared with those of the TGF-β1 absent constructs.

This study developed a bioactive knitted silk-collagen sponge scaffold by incorporation of exogenous SDF-1 alpha, to enable selective migration and homing of cells for in situ tendon regeneration. With in vitro studies, it was observed that CXCR4 gene expression and migration of bone mesenchymal stromal cells and hypo-dermal fibroblasts were more sensitive to exogenous SDF-1 alpha, while expression of tendon repair gene markers by hypo-dermal fibroblasts and Achilles tendon fibroblasts were more sensitive to exogenous SDF-1 alpha. With a rat Achilles tendon injury model, exogenous SDF-1 alpha was shown to reduce infiltration of inflammatory cells and enhance migration of fibroblast-like cells into the scaffold at 4 days and 1 week post-surgery. After 4 weeks, SDF-1 alpha treated tendon had increased expression of tendon repair gene markers and endogenous SDF-1 alpha, exhibited more physiological microstructures with larger diameter collagen fibrils, and had better biomechanical properties than the control group. Hence, our bioactive scaffold improved efficacy of tendon regeneration by increasing the recruitment of fibroblast-like cells, enhancing local endogenous SDF-1 alpha and tendon extracellular matrix production, and decreasing accumulation of inflammatory cells. Incorporation of SDF-1 alpha within a knitted silk-collagen sponge scaffold can therefore be a practical application for tendon tissue engineering.

Tendon is a specific connective tissue composed of parallel collagen fibers. The effect of this tissue-specific matrix orientation on stem cell differentiation has not been investigated. This study aimed to determine the effects of nanotopography on the differentiation of human tendon stem/progenitor cells (hTSPCs) and develop a biomimetic scaffold for tendon tissue engineering. The immuno-phenotype of fetal hTSPCs was identified by flow cytometry. The multipotency of hTSPCs toward osteogenesis, adipogenesis, and chondrogenesis was confirmed. Then, the hTSPCs were seeded onto aligned or randomly-oriented poly (l-lactic acid) nanofibers. Scanning electron micrographs showed that hTSPCs were spindle-shaped and well orientated on the aligned nanofibers. The expression of tendon-specific genes was significantly higher in hTSPCs growing on aligned nanofibers than those on randomly-oriented nanofibers in both normal and osteogenic media. In addition, alkaline phosphatase activity and alizarin red staining showed that the randomly-oriented fibrous scaffold induced osteogenesis, while the aligned scaffold hindered the process. Moreover, aligned cells expressed significantly higher levels of integrin alpha1, alpha5 and beta1 subunits, and myosin II B. In in vivo experiments, the aligned nanofibers induced the formation of spindle-shaped cells and tendon-like tissue. In conclusion, the aligned electrospun nanofiber structure provides an instructive microenvironment for hTSPC differentiation and may lead to the development of desirable engineered tendons.

In this study a 3-D alginate microbead platform was coated with cartilaginous extracellular matrix (ECM) components to emulate chondrogenic microenvironment in vivo for the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). BMSCs were seeded onto the microbead surface and the effect of the modified microbead on BMSC adhesion, proliferation and chondrogenic differentiation was studied, and compared to chondrogenesis in conventional pellet culture. Our results indicated that microbead system promoted BMSC proliferation and protein deposition resulting in the formation of bigger aggregates compared to conventional pellet culture. Analysis of the aggregates indicated that chondroitin sulfate (CS)- and Col2-coated microbeads enhanced the chondrogenic differentiation of hBMSCs, with increasing formation of glycosaminoglycan (GAG) and collagen II deposition in histology, immunohistochemistry and real time PCR analysis. In addition, Col2-coated microbeads resulted in hypertrophic maturation of the differentiated chondrocytes, similar to conventional pellet culture, while CS-coated microbeads were able to retain the pre-hypertrophy state of the differentiated cells. Our result suggested that provision of suitable cartilaginous microenvironment in a 3-D system can promote the chondrogenic differentiation of BMSC and influence the phenotype of resulting chondrocytes. Our microbead system provides an easy method of processing a 3-D alginate system that allows the possibility of scaling up chondrogenic pellet production for clinical application, while the modifiable microbeads also provide an adjustable 3-D platform for the study of co-interaction of ECM and differentiation factors during the stem cell differentiation.

Physical topographic cues from various substrata have been shown to exert profound effects on the growth and differentiation of stem cells due to their niche-mimicking features. However, the biological function of different topographic materials utilized as bio-scaffolds in vivo have not been rigorously characterized. This study investigated the divergent differentiation pathways of mesenchymal stem cells (MSCs) and neo-tissue formation trigged by aligned and randomly-oriented fibrous scaffolds, both in vitro and in vivo. The aligned group was observed to form more mature tendon-like tissue in the Achilles tendon injury model, as evidenced by histological scoring and collagen I immunohistochemical staining data. In contrast, the randomly-oriented group exhibited much chondrogenesis and subsequent bone tissue formation through ossification. Additionally, X-ray imaging and osteocalcin immunohistochemical staining also demonstrated that osteogenesis in vivo is driven by randomly oriented topography. Furthermore, MSCs on the aligned substrate exhibited tenocyte-like morphology and enhanced tenogenic differentiation compared to cells grown on randomly-oriented scaffold. qRT-PCR analysis of osteogenic marker genes and alkaline phosphatase (ALP) staining demonstrated that MSCs cultured on randomly-oriented fiber scaffolds displayed enhanced osteogenic differentiation compared with cells cultured on aligned fiber scaffolds. Finally, it was demonstrated that cytoskeletal tension release abrogated the divergent differentiation pathways on different substrate topography. Collectively, these findings illustrate the relationship between topographic cues of the scaffold and their inductive role in tissue regeneration; thus providing an insight into future development of smart functionalized bio-scaffold design and its application in tissue engineering.

Anterior cruciate ligament (ACL) is difficult to heal after injury due to the dynamic fluid environment of joint. Previously, we have achieved satisfactory regeneration of subcutaneous tendon/ligament with knitted silk–collagen sponge scaffold due to its specific “internal-space-preservation” property. This study aims to investigate the long-term effects of knitted silk–collagen sponge scaffold on ACL regeneration and osteoarthritis prevention. The knitted silk–collagen sponge scaffold was fabricated and implanted into a rabbit ACL injury model. The knitted silk–collagen sponge scaffold was found to enhance migration and adhesion of spindle-shaped cells into the scaffold at 2 months post-surgery. After 6 months, ACL treated with the knitted silk–collagen sponge scaffold exhibited increased expression of ligament genes and better microstructural morphology. After 18 months, the knitted silk–collagen sponge scaffold-treated group had more mature ligament structure and direct ligament-to-bone healing. Implanted knitted silk–collagen sponge scaffolds degraded much more slowly compared to subcutaneous implantation. Furthermore, the knitted silk–collagen sponge scaffold effectively protected joint surface cartilage and preserved joint space for up to 18 months post-surgery. These findings thus demonstrated that the knitted silk–collagen sponge scaffold can regenerate functional ACL and prevent osteoarthritis in the long-term, suggesting its clinical use as a functional bioscaffold for ACL reconstruction.

The cell-cycle inhibitor flavopiridol has been shown to improve recovery from spinal cord injury in animal models. However, the systemic dose of flavopiridol has side-effects and the mechanism of action is not clear. This study aimed to develop a strategy for the local delivery of flavopiridol and investigate its mechanisms of action. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were used for the sustained delivery of flavopiridol. The spinal cord was right-hemisectioned and NPs were delivered into the injury site. Transparent spinal cord technology was used for the three-dimensional observation of anterograde tracing. The results showed that flavopiridol NPs had a sustained release of up to 3 days in vitro. Flavopiridol NPs significantly decreased inflammatory factor synthesis by astrocytes, including TNF-α, IL-1β, and IL-6, while the IL-10 expression was elevated. In vivo study demonstrated that flavopiridol NPs decreased cell-cycle activation, inflammatory expression and glial scarring, and facilitated neuronal survival and regeneration. The cavitation volume was decreased by ~90%. Administration of flavopiridol NPs also improved the motor recovery of injured animals. These findings demonstrated that local delivery of flavopiridol in PLGA NPs improves recovery from spinal cord injury by inhibiting astrocyte growth and inflammatory factor synthesis.

Degradation of proteoglycan is the key early event in the development of osteoarthritis (OA). The aggrecanase ADAMTS-5 has been identified as the major enzyme responsible for the degradation and thus is an attractive therapeutic target for OA. However, currently there is no report on using an ADAMTS-5 inhibition strategy for OA treatment. The present study aimed to investigate the synergic effect of combining an ADAMTS-5 inhibitor (114810) with a hyaluronic acid hydrogel (HAX) for OA therapeutics. Two OA models were induced by surgically creating an osteochondral defect or removing the anterior cruciate ligament (ACL) in Sprague-Dawley rats. Human OA cartilage was obtained from total joint replacement patients. Both human and rat OA cartilage showed marked proteoglycan loss with significantly increased ADAMTS-5 expression. The effectiveness of ADAMTS-5 inhibition by 114810 was confirmed by a cartilage explants assay in vitro, which showed that the 114810 halted the aggrecanase-mediated (374)ARGS neoepitope released from aggrecan induced by IL-1β stimulation. The in vivo effect of ADAMTS-5 inhibition was assessed by the articular injection of HAX with 114810 into OA knee joints. Evaluated eight weeks after injection, 114810 with HAX significantly promoted the in vivo cartilage healing in the osteochondral defect model, and prevented the progression of degenerative changes in the ACL model. Our results confirmed that ADAMTS-5 is an effective target for OA treatment, and the intra-articular injection of an ADAMTS-5 inhibitor within HAX gel could be a promising strategy for OA treatment.

Human embryonic stem cells (hESC) and their differentiated progenies are an attractive cell source for transplantation therapy and tissue engineering. Nevertheless, the utility of these cells for tendon tissue engineering has not yet been adequately explored. This study incorporated hESC-derived mesenchymal stem cells (hESC-MSCs) within a knitted silk-collagen sponge scaffold, and assessed the efficacy of this tissue-engineered construct in promoting tendon regeneration. When subjected to mechanical stimulation in vitro, hESC-MSCs exhibited tenocyte-like morphology and positively expressed tendon-related gene markers (e.g. Collagen type I & III, Epha4 and Scleraxis), as well as other mechano-sensory structures and molecules (cilia, integrins and myosin). In ectopic transplantation, the tissue-engineered tendon under in vivo mechanical stimulus displayed more regularly aligned cells and larger collagen fibers. This in turn resulted in enhanced tendon regeneration in situ, as evidenced by better histological scores and superior mechanical performance characteristics. Furthermore, cell labeling and extracellular matrix expression assays demonstrated that the transplanted hESC-MSCs not only contributed directly to tendon regeneration, but also exerted an environment-modifying effect on the implantation site in situ. Hence, tissue-engineered tendon can be successfully fabricated through seeding of hESC-MSCs within a knitted silk-collagen sponge scaffold followed by mechanical stimulation.

Stress urinary incontinence remains a worldwide problem affecting patients of all ages. Implantation of suburethral sling is the cornerstone treatment. Current slings have inherent disadvantages. This study aims to develop a tissue engineered sling with bone marrow derived mesenchymal stem cell seeded degradable silk scaffold. The mesenchymal stem cells were obtained from Sprague-Dawley rats and were characterized in vitro. Layered cell sheets were formed after two weeks of culture and were labeled with carboxyfluorescein diacetate. Forty female rats were divided into four groups: Group A (n=5) had sham operation; other three groups underwent bilateral proximal sciatic nerve transection and were confirmed with stress urinary incontinence by the leak-point pressure measurement at 4 weeks after operation. Then, Group B (n=5) had no sling placed; Group C (n=15) was treated with a silk sling; and Group D (n=15) was treated with the tissue engineered sling. Histology and the leak-point pressure measurements were done at 4 and 12 weeks after the sling implantation while collagen content and mechanical testing were done at 12 weeks. The results showed that Group B had a significantly lower leak-point pressure (24.0+/-4.2 cmH(2)O) at 4 weeks (P0.05), while Group C (38.0+/-3.3 cmH(2)O) and Group D (36.3+/-3.1 cmH(2)O) almost reached to the normal level shown by Group A (41.6+/-3.8 cmH(2)O) (p0.05). At 12 weeks, tissue engineered sling of group D has higher collagen content (70.84+/-14.49 microg/mg) and failure force (2.436+/-0.192 N) when compared those of Group C (38.94+/-7.05 microg/mg and 1.521+/-0.087 N) (p0.05). Both the silk sling and tissue engineered sling showed convincing functional effects for the treatment of stress urinary incontinence in a rat model. And the better ligament-like tissue formation in the tissue engineered sling suggested potential long-term function.

A composite construct comprising of bone marrow mesenchymal stem cells (BMSCs), plasmid DNA encoding transforming growth factor-beta1 (pDNA-TGF-beta1), fibrin gel and poly (lactide-co-glycolide) (PLGA) sponge was designed and employed to repair articular cartilage defects. To improve the gene transfection efficiency, a cationized chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 9% to BMSCs and showed heterogeneous TGF-beta1 expression in a 10-day culture period in vitro. In vivo culture of the composite constructs was performed by implantation into full-thickness cartilage defects of New Zealand white rabbit joints, using the constructs absence of pDNA-TGF-beta1 or BMSCs as controls. Heterogeneous expression of TGF-beta1 in vivo was detected at 4 weeks, but its level was decreased in comparison with that of 2 weeks. After implantation for 12 weeks, the cartilage defects were successfully repaired by the composite constructs of the experimental group, and the neo-cartilage integrated well with its surrounding tissue and subchondral bone. Immunohistochemical and glycosaminoglycans (GAGs) staining confirmed the similar amount and distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The cartilage special genes expressed in the neo-tissue were closer to those of the normal cartilage. An overall score of 2.83 was obtained according to Wakitani's standard. By contrast, only part of the defects was repaired by the pDNA-TGF-beta1 absence constructs, and no cartilage repair but fibrous tissue was found for the BMSCs absence constructs. Therefore, combination of the PLGA sponge/fibrin gel scaffold with BMSCs and gene therapy is an effective method to restore cartilage defects and may have a great potential for practical applications in the near future.

This study was aimed to develop a new practical ligament scaffold by synergistic incorporation of silk fibers, a knitted structure, and a collagen matrix. The efficacy for ligament tissue engineering was investigated in vitro and in animal models. Cells cultured on a collagen substrate expressed ligament matrix genes at higher levels than those on a silk substrate. The silk scaffold elicited little inflammatory reaction and degraded slowly after subcutaneous implantation in a mouse model. In the rabbit MCL defect model, MCLs treated with a silk+collagen scaffold deposited more collagen, had better mechanical properties, and showed more native microstructure with larger diameter collagen fibrils and stronger scaffold-ligament interface healing than untreated MCLs and those treated with silk scaffolds. These results demonstrated that the knitted silk+collagen sponge scaffold improves structural and functional ligament repair by regulating ligament matrix gene expression and collagen fibril assembly. The findings are the first to highlight the important roles of biomaterials in ligament regeneration biology. Also, the concept of an "internal-space-preservation" scaffold is proposed for the tissue repair under physical loading.