Epidermal-dermal coupled spheroids are important for tissue pattern regeneration in reconstituted skin explant cultures.

Tissue patterning is critical for the development and regeneration of organs. To advance the use of engineered reconstituted skin organs, we study cardinal features important for tissue patterning and hair regeneration. We find they spontaneously form spheroid configurations, with polarized epidermal cells coupled with dermal cells through a newly formed basement membrane. Functionally, the spheroid becomes competent morphogenetic units (CMU) that promote regeneration of tissue patterns. The emergence of new cell types and molecular interactions during CMU formation was analyzed using scRNA-sequencing. Surprisingly, in newborn skin explants, IFNr signaling can induce apical-basal polarity in epidermal cell aggregates. Dermal-Tgfb induces basement membrane formation. Meanwhile, VEGF signaling mediates dermal cell attachment to the epidermal cyst shell, thus forming a CMU. Adult mouse and human fetal scalp cells fail to form a CMU but can be restored by adding IFNr or VEGF to achieve hair regeneration. We find different multi-cellular configurations and molecular pathways are used to achieve morphogenetic competence in developing skin, wound-induced hair neogenesis, and reconstituted explant cultures. Thus, multiple paths can be used to achieve tissue patterning. These insights encourage more studies of "in vitro morphogenesis" which may provide novel strategies to enhance regeneration.

COX2-ATP Synthase Regulates Spine Follicle Size in Hedgehogs.

Skin evolves essential appendages with adaptive patterns that synergistically insulate the body from environmental insults. How similar appendages in different animals generate diversely-sized appendages remain elusive. Here we used hedgehog spine follicles and mouse hair follicles as models to investigate how similar follicles form in different sizes postnatally. Histology and immunostaining show that the spine follicles have a significantly greater size than the hair follicles. By RNA-sequencing analysis, we found that ATP synthases are highly expressed in hedgehog skin compared to mouse skin. Inhibition of ATP synthase resulted in smaller spine follicle formation during regeneration. We also identified that the mitochondrial gene COX2 functions upstream of ATP synthase that influences energy metabolism and cell proliferation to control the size of the spine follicles. Our study identified molecules that function differently in forming diversely-sized skin appendages across different animals, allowing them to adapt to the living environment and benefit from self-protection.

Mechanical force drives the initial mesenchymal-epithelial interaction during skin organoid development.

Rationale: Stem cells self-organize to form organoids that generate mini-organs that resemble the physiologically-developed ones. The mechanism by which the stem cells acquire the initial potential for generating mini-organs remains elusive. Here we used skin organoids as an example to study how mechanical force drives initial epidermal-dermal interaction which potentiates skin organoids to regenerate hair follicles. Methods: Live imaging analysis, single-cell RNA-sequencing analysis, and immunofluorescence were used to analyze the contractile force of dermal cells in skin organoids. Bulk RNA-sequencing analysis, calcium probe detection, and functional perturbations were used to verify that calcium signaling pathways respond to the contractile force of dermal cells. In vitro mechanical loading experiment was used to prove that the stretching force triggers the epidermal Piezo1 expression which negatively regulates dermal cell attachment. Transplantation assay was used to test the regenerative ability of skin organoids. Results: We found that dermal cell-derived contraction force drives the movement of dermal cells surrounding the epidermal aggregates to trigger initial mesenchymal-epithelial interaction (MEI). In response to dermal cell contraction force, the arrangement of the dermal cytoskeleton was negatively regulated by the calcium signaling pathway which further influences dermal-epidermal attachment. The native contraction force generated from the dermal cell movement exerts a stretching force on the adjacent epidermal cells, activating the stretching force sensor Piezo1 in the epidermal basal cells during organoid culture. Epidermal Piezo1 in turn drives strong MEI to negatively regulate dermal cell attachment. Proper initial MEI by mechanical-chemical coupling during organoid culture is required for hair regeneration upon transplantation of the skin organoids into the back of the nude mice. Conclusion: Our study demonstrated that mechanical-chemical cascade drives the initial event of MEI during skin organoid development, which is fundamental to the organoid, developmental, and regenerative biology fields.

Matrix stiffness-dependent STEAP3 coordinated with PD-L2 identify tumor responding to sorafenib treatment in hepatocellular carcinoma.

BACKGROUND: Ferroptosis have been implicated in tumorigenesis, tumor progression, and chemo- and immuno-therapy in cirrhotic hepatocellular carcinoma (HCC), indicating its association with matrix stiffness and clinical benefit of targeting drugs or immune checkpoint inhibitor. Here, we postulated that increased matrix stiffness reduces ferroptosis and impairs tumor immunity by regulating the expression of ferroptosis- and immune-related genes in HCC, which might be a robust predictor of therapeutic efficacy. METHODS: Using publicly available tissue microarray datasets, liver cancer rat model, and clinical specimen, ferroptosis-related differential genes in HCV-infected cirrhotic HCC and its mechanical heterogeneous pattern of expression were screened and identified. Further investigation on the underlying mechanism of matrix stiffness-regulated ferroptosis and the expression of immune mediator were performed. Finally, threshold analysis of HCC cases with sorafenib treatment revealed the value of clinical applications of these potential predictors. RESULTS: STEAP3 was identified as the ferroptosis-related differential genes in HCV-infected cirrhotic HCC. Stiffer matrix decreased STEAP3 in the invasive front area of HCC and the liver cirrhotic tissue. Contrarily, softer matrix induced STEAP3 in the central area of HCC and the normal liver tissue. Immunological correlation of STEAP3 in cirrhotic HCC showed that STEAP3-mediated immune infiltration of CD4+ T and CD8+ T cells, macrophages, neutrophils, and dendritic cells and HCC prognosis, predicting to regulate immune infiltration. Overexpression of STEAP3 induced ferroptosis and inhibited the expression of immune mediator of PD-L2 on a stiff matrix. Especially, the ferroptosis- and immune-related gene predictive biomarker (FIGPB), including STEAP3 and PD-L2, predicts better clinical benefit of sorafenib in HCC patients. CONCLUSIONS: This finding identifies matrix stiffness impairs ferroptosis and anti-tumor immunity by mediating STEAP3 and PD-L2. More importantly, coordinated with PD-L2, matrix stiffness-dependent STEAP3 could be applied as the independent predictors to favorable sorafenib response, and thus targeting it could be a potential diagnosis and treatment strategy for HCC.

Biomechanical Regulatory Factors and Therapeutic Targets in Keloid Fibrosis.

Keloids are fibroproliferative skin disorder caused by abnormal healing of injured or irritated skin and are characterized by excessive extracellular matrix (ECM) synthesis and deposition, which results in excessive collagen disorders and calcinosis, increasing the remodeling and stiffness of keloid matrix. The pathogenesis of keloid is very complex, and may include changes in cell function, genetics, inflammation, and other factors. In this review, we aim to discuss the role of biomechanical factors in keloid formation. Mechanical stimulation can lead to excessive proliferation of wound fibroblasts, deposition of ECM, secretion of more pro-fibrosis factors, and continuous increase of keloid matrix stiffness. Matrix mechanics resulting from increased matrix stiffness further activates the fibrotic phenotype of keloid fibroblasts, thus forming a loop that continuously invades the surrounding normal tissue. In this process, mechanical force is one of the initial factors of keloid formation, and matrix mechanics leads to further keloid development. Next, we summarized the mechanotransduction pathways involved in the formation of keloids, such as TGF-ß/Smad signaling pathway, integrin signaling pathway, YAP/TAZ signaling pathway, and calcium ion pathway. Finally, some potential biomechanics-based therapeutic concepts and strategies are described in detail. Taken together, these findings underscore the importance of biomechanical factors in the formation and progression of keloids and highlight their regulatory value. These findings may help facilitate the development of pharmacological interventions that can ultimately prevent and reduce keloid formation and progression.

Demethylzeylasteral targets lactate by inhibiting histone lactylation to suppress the tumorigenicity of liver cancer stem cells.

Cancer stem cells drive tumor initiation, progression, and recurrence, which compromise the effectiveness of anti-tumor drugs. Here, we report that demethylzeylasteral (DML), a triterpene anti-tumor compound, suppressed tumorigenesis of liver cancer stem cells (LCSCs) by interfering with lactylation of a metabolic stress-related histone. Using RNA sequencing (RNA-seq) and gas chromatography-mass spectrometric (GC-MS) analysis, we showed that the glycolysis metabolic pathway contributed to the anti-tumor effects of DML, and then focused on lactate downstream regulation as the molecular target. Mechanistically, DML opposed the progress of hepatocellular carcinoma (HCC), which was efficiently facilitated by the increase in H3 histone lactylation. Two histone modification sites: H3K9la and H3K56la, which were found to promote tumorigenesis, were inhibited by DML. In addition, we used a nude mouse tumor xenograft model to confirm that the anti-liver cancer effects of DML are mediated by regulating H3 lactylation in vivo. Our findings demonstrate that DML suppresses the tumorigenicity induced by LCSCs by inhibiting H3 histone lactylation, thus implicating DML as a potential candidate for the supplementary treatment of hepatocellular carcinoma.

Mechanic-Driven Biodegradable Polyglycolic Acid/Silk Fibroin Nanofibrous Scaffolds Containing Deferoxamine Accelerate Diabetic Wound Healing.

The extracellular matrix (ECM), comprising of hundreds of proteins, mainly collagen, provides physical, mechanical support for various cells and guides cell behavior as an interactive scaffold. However, deposition of ECM, especially collagen content, is seriously impaired in diabetic wounds, which cause inferior mechanical properties of the wound and further delay chronic wound healing. Thus, it is critical to develop ECM/collagen alternatives to remodel the mechanical properties of diabetic wounds and thus accelerate diabetic wound healing. Here, we firstly prepared mechanic-driven biodegradable PGA/SF nanofibrous scaffolds containing DFO for diabetic wound healing. In our study, the results in vitro showed that the PGA/SF-DFO scaffolds had porous three-dimensional nanofibrous structures, excellent mechanical properties, biodegradability, and biocompatibility, which would provide beneficial microenvironments for cell adhesion, growth, and migration as an ECM/collagen alternative. Furthermore, the data in vivo showed PGA/SF-DFO scaffolds can adhere well to the wound and have excellent biodegradability, which is helpful to avoid secondary damage by omitting the removal process of scaffolds. The finite element analysis results showed that the application of silk fibroin-based scaffolds could significantly reduce the maximum stress around the wound. Besides, PGA/SF-DFO scaffolds induced collagen deposition, re-vascularization, recovered impaired mechanical properties up to about 70%, and ultimately accelerated diabetic wound healing within 14 days. Thus, our work provides a promising therapeutic strategy for clinically chronic wound healing.

MSC-derived immunomodulatory extracellular matrix functionalized electrospun fibers for mitigating foreign-body reaction and tendon adhesion.

Adhesion formation during tendon healing remains a severe problem in clinical practice. Multiple factors contribute to postoperative adhesion formation, and macrophage-driven inflammation is thought to be greatly involved in this process. We hypothesize that reducing macrophage-mediated inflammation in the injured tendon by regulating M1 to M2 macrophage polarization may effectively inhibit adhesion formation. Here, we developed an acellular immunomodulatory biomaterial consisting of an electrospun polycaprolactone/silk fibroin (PCL/SF) composite fibrous scaffold functionalized with mesenchymal stem cell (MSC)-derived extracellular matrix (ECM). To enhance the immunoregulatory potential of MSCs, we performed inflammatory licensing with IFN-γ to obtain immunomodulatory ECM (iECM). Proteomic analyses of MSCs and their secreted ECM components from different culture conditions revealed the MSC-ECM molecular signatures and the potential mechanism of ECM immunoregulation. Then, the immunoregulatory potential of the iECM-modified scaffold was evaluated in vitro and in vivo. Relative to the PCL/SF fibrous scaffold, the iECM-functionalized scaffold facilitated M2 macrophage polarization and inhibited the expression of multiple cytokines (IL-1ß, IL-6, CXCL11, IL-10, IL-1R2, and TGF-ß1) in vitro, strongly suggesting the immunosuppressive ability of iECM derived from inflammatory licensed MSCs. Consistent with the in vitro findings, the results of rat subcutaneous implantation indicated that a markedly lower foreign-body reaction (FBR) was obtained in the PCL/SF-iECM group than in the other groups, as evidenced by thinner fibrotic capsule formation, less type I collagen production and more M2-type macrophage polarization. In the rat Achilles tendon injury model, the PCL/SF-iECM scaffold greatly mitigated tendon adhesion with clear sheath space formation between the tendon and the scaffold. These data highlight the immunomodulatory potential of iECM-functionalized fibrous scaffolds to attenuate FBR by modulating M2 macrophage polarization, thereby preventing tendon adhesion. STATEMENT OF SIGNIFICANCE: Electrospun PCL/SF fibrous scaffolds functionalized with ECM secreted by MSCs stimulated by inflammatory factor IFN-γ was developed that combined physical barrier and immunomodulatory functions to prevent tendon adhesion formation. PCL/SF micro-nanoscale bimodal fibrous scaffolds prepared by emulsion electrospinning possess high porosity and a large pore size beneficial for nutrient transport to promote intrinsic healing; moreover, surface modification with immunomodulatory ECM (iECM) mitigates the FBR of fibrous scaffolds to prevent tendon adhesion. The iECM-functionalized electrospun scaffolds exhibit powerful immunomodulatory potency in vitro and in vivo. Moreover, the iECM-modified scaffolds, as an anti-adhesion physical barrier with immunomodulatory ability, have an excellent performance in a rat Achilles tendon adhesion model. MSC secretome-based therapeutics, as an acellular regenerative medicine strategy, are expected to be applied to other inflammatory diseases due to its strong immunoregulatory potential.

Gradient Biomineralized Silk Fibroin Nanofibrous Scaffold with Osteochondral Inductivity for Integration of Tendon to Bone.

Enthesis injury repair remains a huge challenge because of the unique biomolecular composition, microstructure, and mechanics in the interfacial region. Surgical reconstruction often creates new bone-scaffold interfaces with mismatched properties, resulting in poor osseointegration. To mimic the natural interface tissue structures and properties, we fabricated a nanofibrous scaffold with gradient mineral coating based on 10 × simulated body fluid (SBF) and silk fibroin (SF). We then characterized the physicochemical properties of the scaffold and evaluated its biological functions both in vitro and in vivo. The results showed that different areas of SF nanofibrous scaffold had varying levels of mineralization with disparate mechanical properties and had different effects on bone marrow mesenchymal stem cell growth and differentiation. Furthermore, the gradient scaffolds exhibited an enhancement of integration in the tendon-to-bone interface with a higher ultimate load and more fibrocartilage-like tissue formation. These findings demonstrate that the silk-based nanofibrous scaffold with gradient mineral coating can regulate the formation of interfacial tissue and has the potential to be applied in interface tissue engineering.

Mechanical stretch induces osteogenesis through the alternative activation of macrophages.

For reconstructive surgeons, critically skeletal damage represents a major challenge. Growing evidence indicate that bone repair is dynamically regulated by the mesenchymal stem cell (MSC)-macrophage interaction. Mechanical strain plays a fundamental role in bone repair and regeneration by influencing MSCs differentiation. Recently, a few findings indicate that macrophages may be mechanically sensitive and their phenotype can be regulated, in part, by mechanical cues. However, how macrophages subjected mechanical stretch influence the osteogenic differentiation of MSCs remain unclear. Thus, the purpose of this study is to explore the effect of macrophages stimulated with mechanical stretch on MSCs osteogenesis. By using a coculture system, we discover that macrophages efficiently induce osteogenic differentiation of MSCs under specific stretch conditions. A synergy mechanism between M2 polarization and YAP/BMP2 axis are identified through molecular and genetic analyses. Macrophages are activated by cyclic stretch and polarized to M2 phenotype that produce anti-inflammatory cytokines such as IL-10 and TGF-ß to regulate the local inflammatory microenvironment. Furthermore, mechanical stretch induces YAP activation and nuclear translocation, subsequently regulates downstream BMP2 expression to facilitate MSCs osteogenesis. These findings not only advance our understanding of the complex influence among the mechanical strain, macrophage inflammatory response as well as the osteogenic differentiation of MSCs, but also reveal a control system from mechanical signals to chemical response then to cell behaviors during bone repair and regeneration.

Lysyl oxidase inhibits TNF-α induced rat nucleus pulposus cell apoptosis via regulating Fas/FasL pathway and the p53 pathways.

AIMS: Intervertebral disc degeneration (IVDD) has been regarded as the main cause of low back pain, which affects 80% of adults and still lack effective treatment. In IVDD, nucleus pulposus (NP) cell apoptosis has widely existed. Lysyl oxidase (LOX) has been demonstrated to protect chondrocyte against apoptosis in the TNF-α-treated human chondrocytes. Therefore, in this study, we investigated the anti-apoptosis effect of LOX on TNF-α-treated rat NP cells. MAIN METHODS: Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot analyses were used to detect the expression of LOX in TNF-α-treated rat NP cells. Then, the toxicity of exogenous LOX and its protective effect was evaluated by Cell Counting kit-8 (CCK-8). NP cell apoptosis was evaluated by flow cytometry analysis and TUNEL assay. The regulatory effects of LOX on the expression of extracellular matrix (ECM) molecules in TNF-α-treated rat NP cells were measured by RT-qPCR, western blot, and ELISA analyses. The molecular mechanism of LOX in regulating NP cell apoptosis was investigated by RT-qPCR and western blot analyses. KEY FINDINGS: The expression of LOX in TNF-α-treated rat NP cells was significantly decreased. Exogenous LOX preserved the cell viability, reduced the rate of apoptosis and improved the ECM secretion in TNF-α-treated rat NP cells. Further molecular mechanism investigation showed that LOX inhibited the Fas/FasL and p53 pathways. SIGNIFICANCES: LOX played an anti-apoptotic role in TNF-α-treated rat NP cells which could be a promising reagent in IVDD treatment.

Tumor progression locus 2 (TPL2) in tumor-promoting Inflammation, Tumorigenesis and Tumor Immunity.

Over the years, tumor progression locus 2 (TPL2) has been identified as an essential modulator of immune responses that conveys inflammatory signals to downstream effectors, subsequently modulating the generation and function of inflammatory cells. TPL2 is also differentially expressed and activated in several cancers, where it is associated with increased inflammation, malignant transformation, angiogenesis, metastasis, poor prognosis and therapy resistance. However, the relationship between TPL2-driven inflammation, tumorigenesis and tumor immunity has not been addressed. Here, we reconcile the function of TPL2-driven inflammation to oncogenic functions such as inflammation, proliferation, apoptosis resistance, angiogenesis, metastasis, immunosuppression and immune evasion. We also address the controversies reported on TPL2 function in tumor-promoting inflammation and tumorigenesis, and highlight the potential role of the TPL2 adaptor function in regulating the mechanisms leading to pro-tumorigenic inflammation and tumor progression. We discuss the therapeutic implications and limitations of targeting TPL2 for cancer treatment. The ideas presented here provide some new insight into cancer pathophysiology that might contribute to the development of more integrative and specific anti-inflammatory and anti-cancer therapeutics.

NFAT5 directs hyperosmotic stress-induced fibrin deposition and macrophage infiltration via PAI-1 in endothelium.

Although stress can significantly promote atherosclerosis, the underlying mechanisms are still not completely understood. Here we successfully unveiled that high salt-induced nuclear factor of activated T cells 5 (NFAT5) control the endothelial-dependent fibrinolytic activity and the inflammatory adhesion-related molecules expression through regulation of plasminogen activator inhibitor-1 (PAI-1). We first observed that high salt diets instigated the expression of NFAT5 and PAI-1 in the endothelium which brought about the fibrin deposition and macrophage infiltration in the atherosclerotic arteries of ApoE-/- mice. Overexpression of NFAT5 increased PAI-1-mediated antifibrinolytic activity and activated inflammatory adhesion-related genes in endothelial cells. Knockdown of NFAT5 by siRNA inhibited the expression of PAI-1, antifibrinolytic and adhesive molecules. Moreover, chromatin immunoprecipitation assay demonstrated that high salt intake significantly promoted the binding of NFAT5 to PAI-1 promoter (TGGAATTATTT) in endothelial cells. Our study identified that NFAT5 has great potential to activate the PAI-1-mediated fibrinolytic dysfunction and inflammatory cell adhesion, thus promoting high salt-induced atherosclerosis disease.

Dual-Peptide-Functionalized Nanofibrous Scaffolds Recruit Host Endothelial Progenitor Cells for Vasculogenesis to Repair Calvarial Defects.

Vasculogenesis (de novo formation of vessels) induced by endothelial progenitor cells (EPCs) is requisite for vascularized bone regeneration. However, there exist few available options for promoting vasculogenesis within artificial bone grafts except for exogenous EPC transplantation, which suffers from the source of EPC, safety, cost, and time concerns in clinical applications. This study aimed at endogenous EPC recruitment for vascularized bone regeneration by using a bioinspired EPC-induced graft. The EPC-induced graft was created by immobilizing two bioactive peptides, WKYMVm and YIGSR, on the surface of poly(ε-caprolactone) (PCL)/poliglecaprone (PGC) nanofibrous scaffolds via a polyglycolic acid (PGA)-binding peptide sequence. Remarkable immobilization efficacy of WKYMVm and YIGSR peptides and their sustained release (over 14 days) from scaffolds were observed. In vivo and in vitro studies showed robust recruitment of EPCs, which subsequently contributed to early vasculogenesis and ultimate bone regeneration. The dual-peptide-functionalized nanofibrous scaffolds proposed in this study provide a promising therapeutic strategy for vasculogenesis in bone defect repair.

NFAT5 mediates hypertonic stress-induced atherosclerosis via activating NLRP3 inflammasome in endothelium.

BACKGROUND: How high-salt intake leads to the occurrence of many cardiovascular diseases such as atherosclerosis is a fundamental question in pathology. Here we postulated that high-salt-induced NFAT5 controls the inflammasome activation by directly regulating NLRP3, which mediates the expression of inflammatory- and adhesion-related genes in vascular endothelium, resulting in the formation of atherosclerosis. METHODS: Atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) mice which accumulate cholesterol ester-enriched particles in the blood due to poor lipoprotein clearance capacity were used as the atherosclerosis model in vivo. Cultured endothelial cells (ECs) and monocytes under high-salt condition were used to explore the atheroprone role of the activation of NFAT5-NLRP3 inflammasome in vascular endothelium in vitro. Bioinformatic analysis and chromatin immunoprecipitation assay were used to identify the DNA binding sites of NFAT5 on promoters of NLRP3 and IL-1ß. RESULTS: We first observe that high-salt intake promotes atherosclerosis formation in the aortas of ApoE-/- mice, through inducing the expression of NFAT5, NLRP3, and IL-1ß in endothelium. Overexpression of NFAT5 activates NLRP3-inflammasome and increases the secretion of IL-1ß in ECs partly via ROS. Chromatin immunoprecipitation assay demonstrates that NFAT5 directly binds to the promoter regions of NLRP3 and IL-1ß in endothelial cells subjected to the high-salt environment. CONCLUSIONS: Our study identifies NFAT5 as a new and essential transcription factor that is required for the early activation of NLRP3-inflammasome-mediated endothelium innate immunity, contributing to the formation of atherosclerosis under hypertonic stress induction.

Disordered Mechanical Stress and Tissue Engineering Therapies in Intervertebral Disc Degeneration.

Low back pain (LBP), commonly induced by intervertebral disc degeneration, is a lumbar disease with worldwide prevalence. However, the mechanism of degeneration remains unclear. The intervertebral disc is a nonvascular organ consisting of three components: Nucleus pulposus, annulus fibrosus, and endplate cartilages. The disc is structured to support our body motion and endure persistent external mechanical pressure. Thus, there is a close connection between force and intervertebral discs in LBP. It is well established that with aging, disordered mechanical stress profoundly influences the fate of nucleus pulposus and the alignment of collagen fibers in the annulus fibrosus. These support a new understanding that disordered mechanical stress plays an important role in the degeneration of the intervertebral discs. Tissue-engineered regenerative and reparative therapies are being developed for relieving disc degeneration and symptoms of lower back pain. In this paper, we will review the current literature available on the role of disordered mechanical stress in intervertebral disc degeneration, and evaluate the existing tissue engineering treatment strategies of the current therapies.

Functionalized cell-free scaffolds for bone defect repair inspired by self-healing of bone fractures: A review and new perspectives.

Studies have demonstrated that scaffolds, a component of bone tissue engineering, play an indispensable role in bone repair. However, these scaffolds involving ex-vivo cultivated cells seeded have disadvantages in clinical practice, such as limited autologous cells, time-consuming cell expansion procedures, low survival rate and immune-rejection issues. To overcome these disadvantages, recent focus has been placed on the design of functionalized cell-free scaffolds, instead of cell-seeded scaffolds, that can reduplicate the natural self-healing events of bone fractures, such as inflammation, cell recruitment, vascularization, and osteogenic differentiation. New approaches and applications in tissue engineering and regenerative medicine continue to drive the development of functionalized cell-free scaffolds for bone repair. In this review, the self-healing processes were highlighted, and approaches for the functionalization were summarized. Also, ongoing efforts and breakthroughs in the field of functionalization for bone defect repair were discussed. Finally, a brief summery and new perspectives for functionalization strategies were presented to provide guidelines for further efforts in the design of bioinspired cell-free scaffolds.

Effect of substrate stiffness on hepatocyte migration and cellular Young's modulus.

Hepatic fibrosis progress accompanied by an unbalanced extracellular matrix (ECM) degradation and deposition leads to an increased tissue stiffness. Hepatocytes interplay with all intrahepatic cell populations inside the liver. However, how hepatocytes migration and cellular Young's modulus influenced by the substrate stiffness are not well understood. Here, we established a stiffness-controllable in vitro cell culture model by using a polyvinyl alcohol (PVA) hydrogel that mimicked the same physical stiffness as a fibrotic liver. Three levels of stiffness were used in our experiment that corresponded to the stiffness levels found in normal liver tissue (4.5 kPa), the early (19 kPa) and late stages (37 kPa) of fibrotic liver tissues. Cytoskeleton of hepatocyte was influenced by substrate stiffness. Soft substrate promoted the cellular migration and directionality. The cellular Young's modulus firstly increased and then decreased with increasing substrate stiffness. Integrin-ß1 and ß-catenin expression on cytomembrane were up-regulated and down-regulated with the increase of substrate stiffness, respectively. Our data not only suggested that hepatocytes were sensitive to substrate stiffness, but also suggested that there may be a potential relationship among substrate stiffness, cellular Young's modulus and the dynamic balance of integrin-ß1 and ß-catenin pathways. These results may provide us a new insight in mechanism investigation of mechano-dependent diseases, especially like fibrosis related diseases.

The use of mechano growth factor to prevent cartilage degeneration in knee osteoarthritis.

Previous studies have attached more importance to growth factors in treating cartilage degeneration and osteoarthritis (OA). Here, the capability of mechano growth factor-C24E (MGF) to prevent osteoarthritic cartilage degeneration was evaluated in vitro and in vivo. Using in vitro cultured human OA chondrocytes treated with 10-60-ng/ml MGF for 12 hr, we detected the cell proliferation, migration, and anabolism of OA chondrocytes. The unfolded protein response and the protein characteristic of OA pathology, such as transforming growth factor ß, SMAD family member 3, and hypoxia-inducible factor 2α of OA chondrocytes, were also detected by western blotting. Furthermore, protein kinase RNA-like endoplasmic reticulum kinase was knocked down via small interfering RNA to illuminate the potential mechanism of MGF's treatment of OA. In a rabbit knee joint OA model, cartilage degeneration was inhibited after 2 weeks of treatment with 0.1-10-µg/ml MGF. This study demonstrated that MGF treatment can inhibit the pathological apoptosis of OA chondrocytes and promote the proliferation, migration, and matrix synthesis of the chondrocytes. The results also demonstrate that the degeneration of OA cartilage can be delayed by MGF treatment partially via unfolded protein response regulated by protein kinase RNA-like endoplasmic reticulum kinase and suggest a potential therapeutic application of MGF for OA treatment.