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The application of medical devices to repair skin damage is clinically accepted and natural polymer enjoys an important role in this field, such as collagen or hyaluronic acid, etc. However, the biosafety and efficacy of these implants are still challenged. In this study, a skin damage animal model was prepared by UV-photoaging and recombinant humanized type III collagen (rhCol III) was applied as a bioactive material to implant in vivo to study its biological effect, comparing with saline and uncrosslinked hyaluronic acid (HA). Animal skin conditions were non-invasively and dynamically monitored during the 8 weeks experiment. Histological observation, specific gene expression and other molecular biological methods were applied by the end of the animal experiment. The results indicated that rhCol III could alleviate the skin photoaging caused by UV radiation, including reduce the thickening of epidermis and dermis, increase the secretion of Collagen I (Col I) and Collagen III (Col III) and remodel of extracellular matrix (ECM). Although the cell-material interaction and mechanism need more investigation, the effect of rhCol III on damaged skin was discussed from influence on cells, reconstruction of ECM, and stimulus of small biological molecules based on current results. In conclusion, our findings provided rigorous biosafety information of rhCol III and approved its potential in skin repair and regeneration. Although enormous efforts still need to be made to achieve successful translation from bench to clinic, the recombinant humanized collagen showed superiorities from both safety and efficacy aspects.

Despite the formation of mechanically inferior fibrocartilage, microfracture (MF) still remains the gold standard to repair the articular cartilage defects in clinical settings. To date, although many tissue-engineering scaffolds have been developed to enhance the MF outcome, the clinical outcomes remain inconsistent. Decellularized extracellular matrix (dECM) is among the most promising scaffold for cartilage repair due to its inheritance of the natural cartilage components. However, the impact of dECM from different developmental stages on cellular chondrogenesis and therapeutic effect remains elusive, as the development of native cartilage involves the distinct temporal dependency of the ECM components and various growth factors. Herein, we hypothesized that the immature cartilage dECM at various developmental stages was inherently different, and would consequently impact the chondrogenic potential BMSCs. In this study, we fabricated three different unidirectional collagen-dECM scaffolds sourced from neonatal, childhood, and adolescent rabbit cartilage tissues, and identified the age-dependent biological variations, including DNA, cartilage-specific proteins, and growth factors; along with the mechanical and degradation differences. Consequently, the different local cellular microenvironments provided by these scaffolds led to the distinctive cell morphology, circularity, proliferation, chondrogenic genes expression, and chondrogenesis of BMSCs in vitro, and the different gross morphology, cartilage-specific protein production, and subchondral bone repair when in combination with microfracture in vivo. Together, this work highlights the immature cartilage dECM at different developmental stages that would result in the diversified effects to BMSCs, and childhood cartilage would be considered the optimal dECM source for the further development of dECM-based tissue engineering scaffolds in articular cartilage repair.

Osteoinductivity is a crucial factor to determine the success and efficiency of posterolateral spinal fusion (PLF) by employing calcium phosphate (Ca-P) bioceramics. In this study, three kinds of Ca-P ceramics with microscale to nanoscale gain size (BCP-control, BCP-micro and BCP-nano) were prepared and their physicochemical properties were characterized. BCP-nano had the spherical shape and nanoscale gain size, BCP-micro had the spherical shape and microscale gain size, and BCP-control (BAM®) had the irregular shape and microscale gain size. The obtained BCP-nano with specific nanotopography could well regulate in vitro protein adsorption and osteogenic differentiation of MC3T3 cells. In vivo rabbit PLF procedures further confirmed that nanotopography of BCP-nano might be responsible for the stronger bone regenerative ability comparing with BCP-micro and BCP-control. Collectedly, due to nanocrystal similarity with natural bone apatite, BCP-nano has excellent efficacy in guiding bone regeneration of PLF, and holds great potentials to become an alternative to standard bone grafts for future clinical applications., Graphical abstract Image 1, Highlights • The nanocrystal of porous BCP ceramic spheres is similar to natural bone apatite. • BCP nanoceramics is conducive to protein adsorption and osteogenic differentiation of MC3T3 cells. • Osteoindutivity of BCP ceramics is a crucial factor to determine the sucess and efficiency of PLF. • BCP ceramic spheres with nanotopography hold great potential in clinical PLF applications.

Porous biphasic calcium phosphate bioceramic (BCP) possesses osteoinductivity to induce the osteoblastic commitment of mesenchymal stem cells (MSCs) and ectopic bone formation. However, the underlying mechanism remains enigmatic. We performed a gene array analysis of MSCs cocultured with BCP to screen for candidate osteoinductive modulators. Na+, K+-ATPase (NKA), an ion transporter, therefore was identified as a crucial ion transporter in regulating the osteogenesis of the cells. NKA activator, a polyclonal antibody, enriched the cytomembrane abundance of NKA and lead to an enhanced osteogenic effect of BCP. As indicated in gene array analysis and suggested by co-immunoprecipitation assay, protein phosphatase 2A (PP2A) was elevated by BCP to dephosphorylate NKA and prevent its endocytosis. The inhibition of NKA by ouabain resulted in an adverse effect on osteoinductivity of BCP. We further altered NKA activity in mice implanted with BCP and found that the intensity and incidence of osteoinduction was increased by the NKA activator. We went one step further by investigating the potential of targeting NKA in osteoporotic bone regeneration. Activating NKA upregulated osteogenic gene expression and calcium deposition ability of osteoporotic osteoblasts. Furthermore, activation of NKA in mice ameliorated estrogen-deficiency induced bone loss, in terms of increased bone mass and improved bending strength. With this osteoinductive bioceramic derived ion transporter target, we demonstrate that the activation of NKA has significant potential to revolutionize the regeneration of bone. STATEMENT OF SIGNIFICANCE: In this study, we identified an important role of Na+, K+-ATPase (NKA) have played in osteoinductivity of biphasic calcium phosphate bioceramic (BCP). Furthermore, we demonstrated the therapeutic potential of targeting NKA in osteoporotic bone regeneration. Numerous gene and protein targets to treat osteoporosis were discovered every year, mainly obtained by genomic and proteomic screenings of a large population. In contrast, our study identified an unrevealed bone regenerating target from the upregulated genes induced by an osteoinductive biomaterial. The approach was cost-saving since it did not require a large sample pool. Furthermore, the target derived from this approach was proven to be anabolic. Identification of an anabolic agent holds significant value since most of the current anti-osteoporotic therapies are antiresorptive.

Orthopedic implants with heterogeneous porous structures were known as ideal bone osteointegration. This research introduced the selective laser melting (SLM), finite element analysis (FEA), and a hydrothermal process (HT) for manufacturing a three-level heterogeneous porous structure. The macroporous structure was designed via CAD and micropores were tuned via laser power regulation. A nano-size layer of hydroxyapatite crystals was coated by an HT process. The mechanical properties were reinforced via a core-shell structure with core reinforcement. The existence of micropores and nano-hydroxyapatite coating enhanced the in vitro proliferation of preosteoblasts and osteogenic cellular behaviors of rBMSCs. Thus, the three-level heterogeneous porous titanium implants could inspire researchers with potential clue of cyto-implant interaction mechanism, therefore building ideal orthopedic implants with accelerated osteointegration. STATEMENT OF SIGNIFICANCE: Porous structures of titanium implants play an important role in bone tissue regeneration; The geometrical environment influence cell behaviour and bone tissue ingrowth in all macro-/micro-/nanoscale. In this study, a novel method to fabricate heterogeneous scaffolds and its macro-/micro-/nanoscopic structures were studied. A CAD model was used to obtain the macroscopic structure and the insufficient laser power was introduced for porous microstructure. Therefore, a layer of nano hydroxyapatite was coated via hydrothermal process. Cytoproliferation and cytodifferentiation results indicated that a integrity of regular/irregular, macro-/micro-/nanoscale porous structure had advance in recruiting stem cells and promoting differentiation. This research is beneficial to the development of bone implants with better bone regeneration ability.

The existing approaches for healing mandibular condylar osteochondral defects, which are prevalent in temporomandibular joint disorders (TMD), are sparse and not reparative. To address this, regenerative medicine in situ has transpired as a potential therapeutic solution as it can effectively regenerate composite tissues. Herein, injectable self-crosslinking thiolated hyaluronic acid (HA-SH)/type I collagen (Col I) blend hydrogel and BCP ceramics combined with rabbit bone mesenchymal stem cells (rBMSCs)/chondrocytes were used to fabricate a new bi-layer scaffold to simulate specific structure of rabbit condylar osteochondral defects. The in vitro results demonstrated that the blend hydrogel scaffold provided suitable microenvironment for simultaneously realizing proliferation and chondrogenic specific matrix secretion of both rBMSCs and chondrocytes, while BCP ceramics facilitated rBMSCs proliferation and osteogenic differentiation. The in vivo results confirmed that compared with cell-free implant, the rBMSCs/chondrocytes-loaded bi-layer scaffold could effectively promote the regeneration of both fibrocartilage and subchondral bone, suggesting that the bi-layer scaffold presented a promising option for cell-mediated mandibular condylar cartilage regeneration.

Co-culture of chondrocytes and mesenchymal stem cells (MSCs) represents an effective way to stimulate the chondrogenesis of MSCs and reduce hypertrophy, but the limited donor site supply and the requirement of two-stage operations are among the major barriers of using autologous chondrocytes in clinical. With recent evidence indicated that the chondrogenic effects of the above co-culture mainly lied on the paracrine secretion, and cell membranes also played crucial roles during the chondrocytes-MSCs interaction, we fabricated a multifunctional design of “artificial chondrocytes” that consists of chondrocytes secretome enriched PLGA microparticles with the encapsulation of chondrocytes’ membrane fragments. The artificial chondrocytes had shown a similar diameter and surface electrical charge of natural chondrocytes, with the preserved key chondrocyte membrane surface proteins and sustainedly released chondrogenic cytokines from chondrocytes secretome to extend their effects in vivo. Consequently, the co-culture studies of artificial chondrocytes and bone marrow MSCs had shown the beneficial effects from both chondrocytes secretome and membrane fragments, which also synergistically facilitated the cell proliferation, chondrogenic genes expression, cartilaginous matrix production, and reduced phenotypic hypertrophy in vitro and in vivo. Together, this study has successfully developed the proof-of-concept design of “artificial chondrocytes”, which could potentially conquer many major barriers of using natural chondrocytes and provided a novel synthetic-cell approach to current therapeutical strategies towards the functional regeneration of articular cartilage.

Although in vitro studies have shown that biomaterials and mechanical stimuli can mediate inflammatory responses or regulate osteogenesis of MSCs, the underlying behaviour of the inflammatory response of macrophages on biomaterials mediated by mechanical stimuli, which regulates osteogenesis, is relatively unknown. Thus, it is imperative to explore the role of bionic mechanical stimulation in the biomaterial-mediated inflammatory response of macrophages. In this study, we used osteoinductive biphasic calcium phosphate (BCP) ceramics as the model biomaterial and chose micro-vibration stimulation (MVs) with three variable parameters (frequency, magnitude, and time). Based on orthogonal experiments, nine combinations of MVs parameters were generated, and their effects on the BCP-mediated macrophage inflammatory response were investigated. MVs significantly affected the gene expression and cytokine secretion of macrophages grown on BCP ceramics and further influenced the behaviour of bone marrow mesenchymal stem cells (BMMSCs) in a paracrine manner. Moreover, frequency seemed to be the most dominant factor (compared with magnitude and time) in regulating the inflammatory response of macrophages. The optimal combination of MVs parameters (frequency 10 Hz, magnitude 0.45 g, and time 60 min) could induce a healing-associated M2 phenotype, as evidenced by the downregulated pro-inflammatory gene (Il-1β, and Tnf-α) expression, the upregulated anti-inflammatory gene (Il10) expression, and the inhibited pro-inflammatory cytokine (Il-1β and Tnf-α) secretion of macrophages grown on BCP ceramics, and its conditioned medium (CM) could further promote osteogenic differentiation of BMMSCs. These findings provide valuable insights into the mechanical stimulus-mediated macrophage inflammatory response and osteogenesis in the presence of osteoinductive BCP ceramics and allow accurate evaluation of the biological performance of biomaterials in vitro, in order to optimize bone substitute materials to achieve the desired clinical performance.

Self-crosslinkable and injectable hydrogels were fabricated with collagen type I (Col I) and N-hydroxy sulfosuccinimide activated hyaluronic acid (HA-sNHS) at physiological conditions without any initiators or crosslinkers. The physical properties of hydrogels, such as gelation time, swelling property, degradation property and mechanical property could be regulated by adjusting the substitution degree (DS) of HA-sNHS. Chondrocytes were encapsulated into hydrogels and their proliferation, phenotype maintenance and matrix secretion were characterized. The results demonstrated that chondrocytes in hydrogel Col I/HA-sNHS32% in which the DS of HA-sNHS was 32% secreted more cartilage specific matrix than others. The results of animal experiment demonstrated that hydrogels Col I and Col I/HA-sNHS32% both had good biodegradability and cytocompatibility. This study provided a novel and simple method for fabrication of self-crosslinkable and injectable hydrogels with tunable physical properties. It implied that these hydrogels could find some applications in the fields of cell encapsulation and tissue engineering.

Hyaluronic acid (HA) is widely distributed in the human body, and it is heavily involved in many physiological functions such as tissue hydration, wound repair, and cell migration. In recent years, HA and its derivatives have been widely used as advanced bioactive polymers for bone regeneration. Many medical products containing HA have been developed because this natural polymer has been proven to be nontoxic, noninflammatory, biodegradable, and biocompatible. Moreover, HA-based composite scaffolds have shown good potential for promoting osteogenesis and mineralization. Recently, many HA-based biomaterials have been fabricated for bone regeneration by combining with electrospinning and 3D printing technology. In this review, the polymer structures, processing, properties, and applications in bone tissue engineering are summarized. The challenges and prospects of HA polymers are also discussed.

Injectable hydrogels have attracted increasing attention because of convenient clinical operation, non-invasive surgical procedure and seamless filling of irregular defects. Here, injectable di-self-crosslinking HSMSSA hydrogel was formed via fast thiol/maleimide click chemistry reaction and thiol oxidation reaction as primary and secondary self-crosslinking network, respectively. Molecular weight and precursor concentration significantly affected physichemical properties and biological functions of hydrogels. Although single HSMSSA gel (0.1 M Da, 10 mg/mL) had moderate injectability, preferable mechanical properties and good proliferative ability of chondrocytes in vitro, and could greatly promote cartilaginous tissue formation in vivo, the lack of adhesion sites resulted in an untenable situation in maintaining effective connections among newborn cell clusters. However, the biomimetic injectable di-self-crosslinking blend hydrogel by combing injectable HSMSSA and bioactive Col I had improved resistance to degradation, chondrocytes adhesion and proliferation, especially for multiples ascending genes expression level associated with hyaline cartilage formation and polyproteoglycan secretion, which might be a potential clinical treatment strategy for constructing injectable cartilage repair filler by combining expanded autologous chondrocytes. Statement of Significance An injectable di-self-crosslinking Hyaluronan–Based hydrogel was formed via fast thiol/maleimide click chemistry reaction and thiol oxidation reaction as primary/secondary self-crosslinking network, respectively. Molecular weight and precursor concentration significantly affected physichemical properties and biological functions of the hydrogels. Although this HSMSSA gel (0.1 M Da, 10 mg/mL) had moderate injectability, preferable mechanical properties, and good proliferative ability of chondrocytes in vitro, and could greatly promote cartilaginous tissue formation in vivo, the lack of adhesion sites resulted in ineffective connections among newborn cell clusters. The biomimetic injectable di-self-crosslinking blend hydrogel improved chondrocyte adhesion and proliferation by combined injectable HSMSSA and bioactive Col I, especially for multiple ascending gene expression levels associated with hyaline cartilage formation and polyproteoglycan secretion.

Melanoma is a highly malignant skin tumor which is prone to recurrence and metastasis. Hydroxyapatite nanoparticles (nHAPs) were reported to possess a suppressive effect on proliferation of various tumor cells in vitro. This study aimed to assess in vitro and in vivo anti-tumor ability and biosafety of the nHAPs used in the treatment of melanoma. Three types of nHAPs with different morphology and crystallinity were synthesized. In vitro cell viability and proliferation studies demonstrated that all three types of nHAPs can inhibit viability and proliferation of A375 and SK-MEL-28 melanoma cells in a concentration-dependent manner. In addition, the rod-shape nHAPs with a crystallinity of 45.60% had the most prominent suppressive effect on the two melanoma cells tested. An important positive regulator of G1/S phase transition in cell cycle, Cyclin D1 protein, was reduced by nHAPs treatment in vivo. We further discovered that the migration ability of the nHAPs treated melanoma cells was greatly decreased. RNA sequencing result revealed that melanoma metastasis related genes were down-regulated by nHAPs, including MMP2, MMP14, ITGA9, ITGB3, ITGB4 and S100B. High concentration of nHAPs treatment in melanoma-bearing nude mice showed a strong inhibitory effect on tumor size and weight. Most importantly, hemolysis, electrolyte disturbance or inflammation response was not discovered in the experimental animals from nHAPs treated groups. We proved that the nHAPs synthesized in the current study has a selective effect to suppress melanoma tumor proliferation and was safe with regard to normal cells and tissue.

Recently, researches have confirmed the crucial role of inflammatory response in Ca-P ceramic-induced osteogenesis, however, the underlying mechanism has not yet been fully understood. In this study, BCP and β-TCP ceramics were used as material models to investigate the effect of physicochemical properties on inflammatory response in vitro. The results showed that BCP and β-TCP could support macrophages attachment, proliferation, and spreading favorably, as well as promote gene expressions of inflammatory related cytokines (IL-1, IL-6, MCP-1, and TNF-α) and growth factors (TGF-β, FGF, PDGF, VEGF, IGF, and EGF). BCP showed a facilitating function on the gene expressions earlier than β-TCP. Further coculture experiments performed in vitro demonstrated that the CMs containing various increased cytokines for macrophages pre-culture could significantly promote MSCs osteogenic differentiation, which was confirmed by the gene expressions of osteogenic specific markers and the intracellular OCN product accumulation under the stimulation of BCP and β-TCP ceramics. Further evidence was found from the formation of mineralized nodules in BCM and TCM. In addition, this study showed a concise relationship between Ca-P ceramic induced inflammation and its osteoinductivity that the increased cytokines and growth factors from macrophages could promote MSCs osteogenic differentiation.

While injectable in situ cross-linking collagen hydrogels offer great potential for applying stem cell therapy to regenerate articular cartilage via minimally invasive procedures, the encapsulated cells experience high shear stress during injection, which results in limited cell survival. In this study, surface-modified cellulose nanocrystals (CNCs) have been investigated as green and biocompatible reinforcing agents for collagen hydrogel. Aldehyde-functionalized CNCs (a-CNCs) were produced through a facile one-pot oxidation. A nanocomposite a-CNC/collagen hydrogel cross-linked rapidly by dynamic Schiff base bonds based on a-CNCs and collagen under physiological conditions. The a-CNC/collagen hydrogel exhibited fast shear-thinning, self-healing characteristics, and improved elastic modulus compared with CNC/collagen hydrogel without Schiff base bonds. The a-CNC/collagen hydrogel was then investigated for mesenchymal stem cell (MSC) delivery. MSCs encapsulated in the a-CNC/collagen hydrogel showed high cell viability after extrusion in vitro. Subcutaneous injection of MSCs encapsulated in the a-CNC/collagen hydrogel showed improved implant integrity and higher cell retention. The proposed self-healing collagen-based hydrogel would not only protect cells during injection but also fit into the irregular cartilage defect, thus holding promise in delivering MSCs for cartilage regeneration through minimally invasive procedures.

During the development of natural cartilage, mesenchymal condensation is the starting event of chondrogenesis, and mesenchymal stem cells (MSCs) experienced a microenvironment transition from primarily cell-cell interactions to a later stage, where cell-extracellular matrix (ECM) interactions dominate. Although micromass pellet culture has been developed to mimic mesenchymal condensation in vitro, the molecular mechanism remains elusive, and the transition from cell-cell to cell-ECM interactions has been poorly recapitulated. In this study, we first constructed MSC microspheres (MMs) and investigated their chondrogenic differentiation with functional blocking of N-cadherin. The results showed that early cartilage differentiation and cartilage-specific matrix deposition of MSCs in the group with the N-cadherin antibody were significantly postponed. Next, poly(l-lysine) treatment was transiently applied to promote the expression of N-cadherin gene, CDH2, and the treatment-promoted MSC chondrogenesis. Upon one-day culture in MMs with established cell-cell adhesions, collagen hydrogel-encapsulated MMs (CMMs) were constructed to simulate the cell-ECM interactions, and the collagen microenvironment compensated the inhibitory effects from N-cadherin blocking. Surprisingly, chondrogenic-differentiated cell migration, which has important implications in cartilage repair and integration, was found in the CMMs without N-cadherin blocking. In conclusion, our study demonstrated that N-cadherin plays the critical role in early mesenchymal condensation, and the collagen hydrogel provides a supportive microenvironment for late chondrogenic differentiation. Therefore, sequential presentations of cell-cell adhesion and cell-ECM interaction in an engineered microenvironment seem to be a promising strategy to facilitate MSC chondrogenic differentiation.

Although there have been many reports on the use of crosslinked hyaluronic acid and gelatin derivatives as injectable hydrogels in cartilage tissue engineering, however, almost no reports have analyzed the kinds of bonding intensity that were most conducive for the maintenance of cartilage phenotypes. Herein, the biomimetic composite hydrogels based on thiolated hyaluronic acid and modified gelatin derivatives with physical mixed, weak, and strong bonding intensity were fabricated, wherein the thiolated hyaluronic acid ensured the basic network structure of composite hydrogels, and gelatin derivatives endowed the bioactivity to hydrogels. These physicochemical properties of composite hydrogels implied that strong bonding intensity (HA-GSH) contributed to the maintenance of a more uniform pore structure, and increased the ability of water retention and resistance to degradation. Further immunohistochemical and RT-PCR analyses demonstrated that the HA-GSH hydrogel greatly improved the expression level of the associated cartilage matrix and the possibility of hyaline cartilage formation in comparison to the physically blended HA-Gel gel and weak bonding crosslinked HA-GMA gel. Overall, all results proved that strong bonding intensity of the disulfide bonds in the HA-GSH hydrogel was more beneficial for the proliferation of chondrocytes and the maintenance of the hyaline cartilage phenotype, which might provide valuable inspiration for designing cartilage repair scaffolds.

Reconstruction of osteoporotic bone defects is a clinical problem that continues to inspire the design of new materials. Methods: In this work, bioceramics composed of strontium (Sr)-doped hydroxyapatite (HA) whiskers or pure HA whiskers were successfully fabricated by hydrothermal treatment and respectively named SrWCP and WCP. Both bioceramics had similar three-dimensional (3D) porous structures and mechanical strengths, but the SrWCP bioceramic was capable of releasing Sr under physiological conditions. In an osteoporotic rat metaphyseal femoral bone defect model, both bioceramic scaffolds were implanted, and another group that received WCP plus strontium ranelate drug administration (Sr-Ran+WCP) was studied for comparison. Results: At week 1 post-implantation, osteogenesis coupled blood vessels were found to be more common in the SrWCP and Sr-Ran+WCP groups, with substantial vascular-like structures. After 12 weeks of implantation, comparable to the Sr-Ran+WCP group, the SrWCP group showed induction of more new bone formation within the defect as well as at the implant-bone gap region than that of the WCP group. Both the SrWCP and Sr-Ran+WCP groups yielded a beneficial effect on the surrounding trabecular bone microstructure to resist osteoporosis-induced progressive bone loss. While an abnormally high blood Sr ion concentration was found in the Sr-Ran+WCP group, SrWCP showed little adverse effect. Conclusion: Our results collectively suggest that the SrWCP bioceramic can be a safe bone substitute for the treatment of osteoporotic bone defects, as it promotes local bone regeneration and implant osseointegration to a level that strontium ranelate can achieve.

Surgical resection and chemotherapy are routinely performed for triple-negative breast cancer (TNBC) because it is insensitive to endocrine therapy and molecular targeted therapy. Here, the optimal surface charge (-28 mV) and particle size (51 nm) enabled the acid-labile hyaluronic acid pullulan prodrug (HPP)-doxorubicin (Dox)/lapatinib (Lap) conjugate to circulate in the blood for a lengthy period of time and enhance the electron paramagnetic resonance effect, while the targeted molecule hyaluronic acid accelerated CD44 receptor-mediated 4T1 cell internalization. The inefficient anti-proliferation capability of Lap increased more than 10-fold after sensitization of Dox to metastatic 4T1 cells, while cellular uptake significantly increased, and cell viability dramatically decreased to nearly 20% of the free Dox group. Furthermore, HPP-Dox/Lap more effectively inhibited lateral mobility, vertical migration, and invasion ability of 4T1 cells. The ex vivo biodistribution of representative Dox indicated that Lap obviously facilitated the intratumoral infiltration and accumulation. The in vivo research revealed that there were overwhelming advantages in using HPP-Dox/Lap to inhibit tumor growth, progression, and lung metastasis even at a low dosage (1 mg kg-1), and it decreased postoperative recurrence and pulmonary metastatic nodules. Because of the excellent biosafety and visible therapeutic effect on the 4T1 metastasis and recurrence model, there is great potential value for HPP-Dox/Lap to be used to treat metastatic TNBC.

The injectable composite hydrogel based on collagen and hyaluronic acid provided a bionic three-dimensional microenvironment and mimetic natural extracellular matrix (ECM) for the growth of cells in vivo and has been widely researched and developed for cartilage tissue engineering. Here, a novel injectable bionic hydrogel with hybrid covalent/noncovalent network derived from covalent conjugation of HA-SH and noncovalent supramolecular self-assembly of BPAA-AFF-OH short peptide was fabricated to overcome the collagen immunogenicity of animal origin and effectively maintain its biological function. Moreover, through optimizing the network structure and polymer composition, the bionic HS5FFAB5 hydrogel presented a reliable mechanical strength which depended on the highly integrated fiber structure between HA-SH and FFAB-AFF-OH molecules. The results in vitro and in vivo proved that HA-SH could provide a fundamental frame structure, while the supramolecular hydrogels could reinforce this structure via hydrogen bonds and hydrophilic/hydrophobic interactions, and endow bionic hydrogels with more abundant cell adhesion sites. The bionic composite hydrogel could improve the cell adhesion and proliferation when compared to HA-SH hydrogel, and enhanced chondrogenic related gene expression and matrix secretion by three-dimensional co-cultured in vitro and subcutaneous implantation in vivo, which further promoted phenotypic maintenance of hyaline cartilage. This bionic hydrogel with a hybrid covalent/noncovalent network is supposed to have potential application prospects in cartilage regeneration.

The self-healing capacity of cartilage was limited due to absence of vascular, nervous and lymphatic systems. Although many clinical treatments have been used in cartilage defect repair and shown a promising repair result in short term, however, regeneration of complete zonal structure with physiological function, reconstruction cartilage homeostasis and maintaining long-term repair was still an unbridgeable chasm. Cartilage has complex zonal structure and multiple physiological functions, especially, superficial and calcified cartilage played an important role in keeping homeostasis. To address this hurdle of regenerating superficial and calcified cartilage, injectable tissue-induced type I collagen (Col I) hydrogel-encapsulated BMSCs was chosen to repair cartilage damage. After 1 month implantation, the results demonstrated that Col I gel was able to induce BMSCs differentiation into chondrocytes, and formed hyaline-like cartilage and the superficial layer with lubrication function. After 3 months post-surgery, chondrocytes at the bottom of the cartilage layer would undergo hypertrophy and promote the regeneration of calcified cartilage. Six months later, a continuous anatomical tidemark and complete calcified interface were restored. The regeneration of neo-hyaline cartilage was similar with adjacent normal tissue on the thickness of the cartilage, matrix secretion, collagen type and arrangement. Complete multilayer zonal structure with physiological function remodeling indicated that BMSCs-assisted injectable Col I hydrogel could reconstruct cartilage homeostasis and maintain long-term therapeutic effect.

The bone regenerative scaffold with the tailored degradation rate matching with the growth rate of the new bone is essential for adolescent bone repair. To satisfy these requirement, we proposed bone tissue scaffolds with controlled degradation rate using osteoinductive materials (Ca-P bioceramics), which is expected to present a controllable biodegradation rate for patients who need bone regeneration. Physicochemical properties, porosity, compressive strength and degradation properties of the scaffolds were studied. 3D printed Ca-P scaffold (3DS), gas foaming Ca-P scaffold (FS) and autogenous bone (AB) were used in vivo for personalized beagle skull defect repair. Histological results indicated that the 3DS was highly vascularized and well combined with surrounding tissues. FS showed obvious newly formed bone tissues. AB showed the best repair effect, but it was found that AB scaffolds were partially absorbed and degraded. This study indicated that the 3D printed Ca-P bioceramics with tailored biodegradation rate is a promising candidate for personalized skull bone tissue reconstruction.

Coculture models have long been used in cell and tissue engineering studies. The direct contact model normally offers excellent cell-cell interactions, but difficult to separate the two cell types after coculture. The non-contact model offers a good cell separation, but the long cell-cell distance limited the cell interactions. This study proposed a conceptual 3D coculture model based on collagen hydrogel microspheres (CHMs) with removable superparamagnetic alginate coating (Alg-Mag-CHMs) to offer a minimal distance and easy separation. The Alg-Mag-CHMs fabrication was achieved using layer-by-layer encapsulation. The thickness could be tailored by applying different numbers of coating layers. Alg-Mag-CHMs had a denser alginate hydrogel shell, which prevented the risk of cell leakage. After mixture, Alg-Mag-CHMs could be rapidly separated in magnet field and the coating could be easily washed off. This study presented proof of concept data of a non-contact 3D coculture model that offered many useful features.

Hydrogels, which provide three-dimensional (3D) niches for encapsulating bone marrow mesenchymal stem cells (BMSCs), are becoming a promising tissue engineering solution for chondrogenic differentiation of BMSCs. However, it remains a challenge to design a hydrogel material for effective chondrogenesis of BMSCs because of the complexity of cartilage ECM and cell-matrix interactions. Thus far, various studies have shown the physical-chemical cues of hydrogel materials to impact BMSCs chondrogenesis, but the design of the 3D network microstructure of the hydrogel to induce BMSCs chondrogenesis is still far from optimized. In this study, we successfully prepared two types of collagen hydrogels, namely, the fibrous network and porous network, with the same chemical composition and similar mechanical strength but with two distinct network microstructures. The two different network microstructures significantly influenced mass transfer, protein adsorption, degradability, and contraction of the collagen hydrogels. Moreover, the cells presented distinct proliferation and morphology in the two hydrogels, which consequently modulated chondrogenic differentiation of BMSCs derived from rat. Collagen hydrogels with a fibrous network promoted more chondrogenic differentiation of BMSCs without additional growth factors in vitro and subcutaneous implantation in vivo than those with a porous network. Moreover, fibrous network resulted in less ECM calcification than porous network. However, the fibrous network could not prevent hypertrophy of the chondrogenic cells induced by BMSCs. Overall, these results revealed that the 3D network microstructure of a hydrogel was a key design parameter for the chondrogenic differentiation of BMSCs. STATEMENT OF SIGNIFICANCE: Hydrogels had been used to induce the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in cartilage tissue engineering, but the key design parameters remain unoptimized. This was mainly due to the different material properties including composition, strength, and microstructure, which would interplay with each other and result in difficulties to investigate the effects for one factor. In this study, we fabricated two collagen hydrogels with the same chemical composition and mechanical strength, but two distinct network microstructures. The effects of the two network microstructures on the chondrogenic differentiation of BMSCs were investigated by in vitro and in vivo assays. The results highlight the effects of network microstructures and provide important information about optimizing the design of future hydrogels in cartilage tissue engineering.

Bone defect and osteoporosis are common in clinic which are seriously harmful for public health. Bionic bone tissue engineering scaffolds are very important for bone tissue repair and reconstruction. In this study, different bionic bone tissue engineering scaffolds were constructed by computer-aided design and fabricated by selected laser melting. Novel porous structures were designed by using parameterization modeling. The accurate models with key characteristics such as porosity and the mechanical property of scaffolds were studied. Compared with the designed model, the error of the selective laser melting (SLM) printed scaffold porosity was less than 2.73%. The mechanical properties of the prepared scaffold can be calculated by finite element analysis of 3D models, and the mechanical properties of the 3D printed samples were consistent with the model design. Through the design, manufacture, characterization and evaluation of the scaffold porous structures, the parametric modeling of porous titanium bone tissue engineering scaffold with good mechanical and biological properties was realized. Optimized design and precisely manufactured implants are very important for bone tissue repair and reconstruction.

Although a promising strategy, the mesenchymal stem cell (MSC)-based therapy of cartilage defects is sometimes accompanied with chronic inflammation during the remodeling status, which may hinder cartilage regeneration. During this process, the inflammatory cytokine tumor necrosis factor α (TNFα) plays an important role and may be a potential target. In this study, we investigated the effect of Tnfα RNA interference by introducing a functional and highly safe carbon dot (CD)-SMCC nanovector synthesized by bioconjugation of CDs with a protein crosslinker, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), as the vehicle of the silenced TNFα (siTnfα) on chondrogenesis of MSCs. The results showed that CD-SMCC displayed intense fluorescence with well-dispersed and positively charged properties, which favored effective binding and delivering of siTnfα into the MSCs. CD-SMCC-siTnfα nanoformula also exhibited considerably high transfection efficiency and nearly no cytotoxicity, which is preferred over commercial polyethyleneimine. Interference of Tnfα by CD-SMCC-siTnfα markedly promoted the chondrogenesis of MSCs, as indicated by upregulating cartilage-specific markers. Furthermore, in vivo exploration indicated that CD-SMCC-siTnfα transfected MSCs accelerated cartilage regeneration. In conclusion, this study demonstrated that in combination with the novel CD-SMCC nanovector, targeting Tnfα may facilitate stem cell-based therapy of cartilage defects. Stem Cells Translational Medicine 2019;8:724&736

Collagen is a promising material for tissue engineering, but the poor mechanical properties of collagen hydrogels, which tend to cause contraction under the action of cellular activity, make its application challengeable. In this study, the amino group of type I collagen (Col I) was modified with methacrylic anhydride (MA) and the photo-crosslinkable methacrylate anhydride modified type I collagen (CM) with three different degrees of substitution (DS) was prepared. The physical properties of CM and Col I hydrogels were tested, including micromorphology, mechanical properties and degradation properties. The results showed that the storage modulus and degradation rate of hydrogels could be adjusted by changing the DS of CM. In vitro, chondrocytes were seeded into these four groups of hydrogels and subjected to fluorescein diacetate/propidium iodide (FDA/PI) staining, cell counting kit-8 (CCK-8) test, histological staining and cartilage-related gene expression analysis. In vivo, these hydrogels encapsulating chondrocytes were implanted subcutaneously into nude mice, then histological staining and sulfated glycosaminoglycan (sGAG)/DNA assays were performed. The results demonstrated that contraction of hydrogels affected behaviors of chondrocytes, and CM hydrogels with suitable DS could resist contraction of hydrogels and promote the secretion of cartilage-specific matrix in vitro and in vivo.

Articular cartilage has very poor intrinsic healing ability and its repair remains a significant clinical challenge. To promote neocartilage regeneration, we fabricated two collagen (Col) scaffolds functionalized with a porcine decellularized extracellular matrix (dECM) in the forms of particle and solution named pE-Col and sE-Col, respectively. Their differences were systematically compared, including the biochemical compositions, scaffold properties, cell-material interactions, and in situ cartilage regeneration. While it is demonstrated that both forms of dECM could enhance the cell recruitment, proliferation, and chondrogenesis of bone marrow stem cells (BMSCs) in vitro, better performance was seen in the sE-Col group, which could quickly provide a more favorable chondrogenic microenvironment for endogenous BMSCs. The superiority of sE-Col was also proved by our in vivo study, which showed that the sE-Col scaffold achieved better structural hyaline-like neocartilage formation and subchondral bone repair compared to the pE-Col scaffold, according to the gross morphology, biological assessment, and micro-CT imaging analysis. Together, this study suggests that the sE-Col scaffold holds great potential in developing the one-step microfracture-based strategy for cartilage repair and also reminds us that despite dECM being a promising biomaterial in tissue engineering, the optimization of the proper processing methodology would be a crucial consideration in the future design of dECM-based scaffolds in articular cartilage regeneration.

Achieving chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) successfully is crucial for cartilage regeneration. To date, various hydrogels with different chemical microenvironment have been used to modulate chondrogenic differentiation of BMSCs, especially collagen and hyaluronic acid hydrogel. However, the chondro-inductive ability of collagen and hyaluronic acid hydrogel has not been evaluated yet and the different chemical and physical microenvironment of these two hydrogels increase the difficulty of comparison. In this study, three different hydrogels based on collagen and hyaluronic acid (self-assembled collagen hydrogel (Col), self-assembled collagen hydrogel cross-linked with genipin (Cgp), and methacrylated hyaluronic acid hydrogel (HA)) were prepared and their chondro-inductive ability on the encapsulated BMSCs was evaluated. Col and Cgp have the same chemical composition and similar microstructure, but are different from HA, while Cgp and HA hydrogels have the same mechanical strength. It was found that chemical and physical microenvironments of the hydrogels combined to influence cell condensation. Thanks to cell condensation was more likely to occur in collagen hydrogels in the early stage, the cartilage-induced ability was in the order of Col > Cgp > HA. However, the severe shrinkage of Col and Cgp resulted in no enough space for cell proliferation within hydrogels in the later stage. In contrast, relatively stable physical microenvironment of HA helped to maintain continuous production of cartilage-related matrix in the later stage. Overall, these results revealed that the chondro-inductive ability of collagen and hyaluronic acid hydrogel with different chemical and physical microenvironment cannot be evaluated by a particular time period. However, it provided important information for optimization and design of the future hydrogels towards successful repair of articular cartilage.

Supramolecular hydrogel composed of aromatic short peptide gelator was an attractive biomaterial owing to its simple and convenient synthetic route, nano-fibrillar microstructure resembling natural collagen fibers and intelligent response to external stimulus. Herein, stimuli-responsive biphenyl-tripeptide supramolecular hydrogels was prepared to simulate extracellular matrix scaffolds by temperature switch, ion induction and pH switch. The amino acid arrangement substantially affected gelation behavior, only BPAA-βAFF and BPAA-FFβA could form nanostructured supramolecular hydrogels with 8-10 nm nanotubes or nanofibers by potential intermolecular hydrogen bond interactions and π-π stacking. The minimum gelation concentration (MGC) and maximum storage modulus were 0.4 mM (0.023 wt%) and around 8.2 KPa. The two supramolecular hydrogels could support adhesion and proliferation of L929 cells. Moreover, the BPAA-βAFF hydrogel promoted proliferation and ECM secretion of chondrocytes in vitro, and facilitate the phenotype maintenance of hyaline cartilage. All the results demonstrated that BPAA-βAFF hydrogel hold great potential application prospects in cartilage tissue engineering. Statement of Significance Diphenylalanine was served as a core segment conjugating with 4-biphenylacetic acid (BPAA) to produce biphenyl-tripeptide compounds with transforming amino sequence, and multiple external stimuli was applied to study the gelation properties of the aromatic short peptide gelators. “FF” brick (phenylalanine-phenylalanine) was crucial for formation of fibrous supramolecular hydrogels. Meanwhile, the sequence of amino acids arrangement also had an essential effect on the gelation behavior. Optimal BPAA-βAFF with ultra-low minimum gelation concentration (0.4 mM, about 0.023 wt%) and similar microstructure to extracellular matrix (ECM) of nature cartilage tissue could promote the proliferation and ECM secretion of chondrocytes in vitro, and facilitate the formation of hyaline cartilage.

The regeneration of bone defects in osteoporotic patients presents a big challenge. Icariin (Ica), which is the main active component of traditional Chinese medicine - herba epimedii, has been widely loaded to scaffolds for the treatment of bone fracture and osteoporosis. To increase drug loading capacity, and meanwhile achieve a stable and safe drug delivery, in present study, a novel icariin derivative - thiolated icariin (Ica-SH) molecule with good cytocompatibility and bioactivity was incorporated into biphasic calcium phosphate (BCP) ceramic scaffold through Ica-SH intermolecular disulfide bonds (abbr., Ica-SH/BCP) to form film coating. Compared with Ica-loaded BCP scaffolds via physical adsorption (abbr., Ica/BCP), the formation of Ica-SH intermolecular disulfide bonds in Ica-SH/BCP scaffolds led to a decreased real-time drug release concentration, and a reduced drug release rate. The released drug from Ica-SH/BCP scaffolds could promote the proliferation, migration, and osteoblastic differentiation of ovariectomized rat bone mesenchymal stem cells (OVX-rBMSCs), and up-regulate the angiogenic gene expression in human umbilical vein endothelial cells (HUVECs) in vitro. The OVX-rat femoral condyle defect model also demonstrated that Ica-SH/BCP scaffolds promoted a superior bone regeneration in osteoporotic animals than BCP ones. These findings suggest that Ica-SH is a promising drug, which can be facilely incorporated into a variety of scaffolds for repairing bone defects of patients, in particular the ones with osteoporosis.

Microfracture surgery remains the most popular treatment for articular cartilage lesions in the clinic, but often leads to the formation of inferior fibrocartilage tissue and damage to subchondral bone. To overcome these problems, extracellular matrix (ECM) scaffolds derived from decellularized natural cartilaginous tissues were introduced and showed excellent biological properties to direct the differentiation of bone marrow stem cells. However, besides the limited allogenic/allogenic supply and the risk of disease transfer from xenogeneic tissues, the effectiveness of ECM scaffolds always varied with a high variability of natural tissue quality. In this study, we developed composite scaffolds functionalized with a cell-derived ECM source, namely, bionic cartilage acellular matrix microspheres (BCAMMs), that support the chondrogenic differentiation of bone marrow cells released from microfracture. The scaffolds with BCAMMs at different developmental stages were investigated in articular cartilage regeneration and subchondral bone repair. Compared to microfracture, the addition of cell-free BCAMM scaffolds has demonstrated a great improvement of regenerated cartilage tissue quality in a rabbit model as characterized by a semi-quantitative analysis of cells, histology and biochemical assays as well as micro-CT images. Moreover, the variation in ECM properties was found to significantly affect the cartilage regeneration, highlighting the challenges of homogenous scaffolds in working with microfracture. Together, our results demonstrate that the biofunctionalized BCAMM scaffold with cell-derived ECM shows great potential to combine with microfracture for clinical translation to repair cartilage defects.

Although platelet-rich plasma (PRP) plays a significant role in the orthopedic clinical application, it still faces two major problems, namely, uncontrollable factors release, frequent preparation and extraction processes as well as the inconvenient form of usage. To overcome these shortcomings, freeze-dried PRP (LyPRP) was encapsulated into bioactive Col I hydrogel to induce osteogenic differentiation of rabbit bone marrow mesenchymal stem cells (rBMSCs). And PRP/Col І composite hydrogel was prepared as a control. Compared with Col І hydrogel, the introduction of platelets significantly improved the mechanical properties of hydrogels. Meanwhile, platelets were evenly distributed in the composite hydrogels network. The sustainable release of related factors in the composite hydrogels could last for more than 14 days to maintain its long-term biological activity. Further cell experiments confirmed that PRP and LyPRP could effectively alleviate the contraction of collagen hydrogel in vitro, and promote the adhesion, proliferation and osteogenesis differentiation of rBMSCs. The results of osteogenic gene expression indicated that the 10% LyPRP/Col І composite hydrogel could facilitate the early expression of BMP-2 and late osteogenic associated protein formation with higher expression of alkaline phosphatase and Osteocalcin (OCN). These results might provide new insights for the clinical application of 10% LyPRP/Col І composite hydrogel as practical bone repair injection.

A biomimetic PEKK implant was developed to promote osteoporotic bone regeneration and delay adjacent bone loss., Osteoporotic fractures are prevalent in society, and their incidence appears to be increasing as the worldwide population ages. However, conventional bone repair materials hardly satisfy the requirements for the repair of pathological fractures. Here, we developed a biomimetic polyetherketoneketone scaffold with a functionalized strontium-doped nanohydroxyapatite coating for osteoporotic bone defect applications. The scaffold has a hierarchically porous architecture and mechanical strength similar to that of osteoporotic trabecular bone. In vitro and in vivo studies demonstrated that the scaffold could promote osteoporotic bone regeneration and delay adjacent bone loss via regulating both osteoblasts and osteoclasts. In addition, the correlations between multiple preimplantation and postimplantation parameters were evaluated to determine the potential predictors of in vivo performance of the material. The current work not only develops a promising candidate for osteoporotic bone repair but also provides a viable approach for designing other functional biomaterials and predicting their translational value.

For reversing the treatment failure in P-glycoprotein (P-gp)-associated MDR (multidrug resistance) of breast cancer, a high dose of Lapatinib (Lap), a substrate of breast cancer-resistant protein, was encapsulated into safe and effective acid-cleavable polysaccharide-doxorubicin (Dox) conjugates to form targeted HPP-Dox/Lap nanoparticles with an optimal drug ratio and appropriate nanosize decorated with oligomeric hyaluronic acid (HA) for specially targeting overexpressed CD44 receptors of MCF-7/ADR. The markedly increased cellular uptake and the strongest synergetic cytotoxicity revealed the enhanced reversal efficiency of HPP-Dox/Lap nanoparticles with reversal multiples at 29.83. This was also verified by the enhanced penetrating capacity in multicellular tumor spheroids. The reinforced Dox retention and substantial down-regulation of P-gp expression implied the possible mechanism of MDR reversal. Furthermore, the efficient ex vivo accumulation and distribution of nanoparticles in the tumor site and the high tumor growth inhibition (93%) even at a lower dosage (1 mg/kg) as well as lung metastasis inhibition in vivo with negligible side effects revealed the overwhelming advantages of targeted polysaccharide nanoparticles and Lap-sensitizing effect against drug-resistant tumor. The development of an efficient and nontoxic-targeted polysaccharide delivery system for reversing MDR by synergistic therapy might provide a potential clinical application value.

Scaffolds with a biomimetic hierarchy micro/nanoscale pores play an important role in bone tissue regeneration. In this study, multilevel porous calcium phosphate (CaP) bioceramic orthopedic implants were constructed to mimic the micro/nanostructural hierarchy in natural wood. The biomimetic hierarchical porous scaffolds were fabricated by combining three-dimensional (3D) printing technology and hydrothermal treatment. The first-level macropores (∼100-600 μm) for promoting bone tissue ingrowth were precisely designed using a set of 3D printing parameters. The second-level micro/nanoscale pores (∼100-10,000 nm) in the scaffolds were obtained by hydrothermal treatment to promote nutrient/metabolite transportation. Micro- and nanoscale-sized pores in the scaffolds were recognized as in situ formation of whiskers, where the shape, diameter, and length of whiskers were modulated by adjusting the components of calcium phosphate ceramics and hydrothermal treatment parameters. These biomimetic natural wood-like hierarchical structured scaffolds demonstrated unique physical and biological properties. Hydrophilicity and the protein adsorption rate were characterized in these scaffolds. In vitro studies have identified micro/nanowhisker coating as potent modulators of cellular behavior through the onset of focal adhesion formation. In addition, histological results indicate that biomimetic scaffolds with porous natural wood hierarchical pores exhibited good osteoinductive activity. In conclusion, these findings combined suggested that micro/nanowhisker coating is a critical factor to modulate cellular behavior and osteoinductive activity.

Mechanical signals have been played close attention to regulate chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In this study, dynamic mechanical loading simulation with natural frequencies and intensities were applied to the 3D cultured BMSCs–collagen scaffold constructs. We investigated the effects of dynamic mechanical loading on cell adhesion, uniform distribution, proliferation, secretion of extracellular matrix (ECM) and chondrogenic differentiation of BMSCs–collagen scaffold constructs. The results indicated that dynamic mechanical loading facilitated the BMSCs adhesion, uniform distribution, proliferation and secretion of ECM with a slight contraction, which significantly improved the mechanical strength of the BMSCs–collagen scaffold constructs for better mimicking the structure and function of a native cartilage. Gene expression results indicated that dynamic mechanical loading contributed to the chondrogenic differentiation of BMSCs with higher levels of AGG, COL2A1 and SOX9 genes, and prevented of hypertrophic process with lower levels of COL10A1, and reduced the possibility of fibrocartilage formation due to down-regulated COL1A2. In conclusion, this study emphasized the important role of dynamic mechanical loading on promoting BMSCs chondrogenic differentiation and maintaining the cartilage phenotype for in vitro reconstruction of tissue-engineered cartilage, which provided an attractive prospect and a feasibility strategy for cartilage repair.

Background Treatment for melanoma is a challenging clinical problem, and some new strategies are worth exploring. Purpose The objective of this study was to investigate the in vitro and in vivo anti-melanoma effects of hydroxyapatite nanoparticles (HANPs) and discuss the involved material factors. Materials and methods Five types of HANPs, ie, HA-A, HA-B, HA-C, HA-D, and HA-E, were prepared by wet chemical method combining with polymer template and appropriate post-treatments. The in vitro effects of the as-prepared five HANPs on inhibiting the viability of A375 melanoma cells and inducing the apoptosis of the cells were evaluated by Cell Counting Kit-8 analysis, cell nucleus morphology observation, flow cytometer, and PCR analysis. The in vivo anti-melanoma effects of HANPs were studied in the tumor model of nude mice. Results The five HANPs had different physicochemical properties, including morphology, size, specific surface area (SSA), crystallinity, and so on. By the in vitro cell study, it was found that the material factors played important roles in the anti-melanoma effect of HANPs. Among the as-prepared five HANPs, HA-A with granular shape, smaller size, higher SSA, and lower crystallinity exhibited best effect on inhibiting the viability of A375 cells. At the concentration of 200 μg/mL, HA-A resulted in the lowest cell viability (34.90%) at day 3. All the HANPs could induce the apoptosis of A375 cells, and the relatively higher apoptosis rates of the cells were found in HA-A (20.10%) and HA-B (19.41%) at day 3. However, all the HANPs showed no inhibitory effect on the viability of the normal human epidermal fibroblasts. The preliminary in vivo evaluation showed that both HA-A and HA-C could delay the formation and growth speed of melanoma tissue significantly. Likely, HA-A exhibited better effect on inhibiting the growth of melanoma tissue than HA-C. The inhibition rate of HA-A for tumor tissue growth reached 49.1% at day 23. Conclusion The current study confirmed the anti-melanoma effect of HANPs and provided a new idea for the clinical treatment of melanoma.

The differentiation of bone marrow mesenchymal stem cells (BMSCs) into functional chondrocytes is crucial for successful cartilage tissue engineering. Since the extracellular matrix (ECM) microenvironment can regulate the behaviours of BMSCs and guide their differentiation, it is important to simulate the natural cartilage ECM to induce the chondrogenesis of BMSCs. As the most abundant protein in the ECM, collagen hydrogels were found to provide a structural and chemical microenvironment for natural cartilage, and regulate the chondrogenic differentiation of BMSCs. However, as the negatively charged ECM microenvironment is crucial for chondrogenesis and homeostasis within cells in cartilage tissue, the electrical properties of collagen hydrogels need to be further optimized. In this study, three collagen hydrogels with different electrical properties were fabricated using methacrylic anhydride (MA) and succinic anhydride (SA) modification. The collagen hydrogels had a similar composition, storage modulus and integral triple helix structure of collagen, but their different negatively charged microenvironments significantly impacted the hydrophilicity, protein diffusion and binding, and consequently influenced BMSC adhesion and spreading on the surface of the hydrogels. Moreover, the BMSCs encapsulated in the collagen hydrogels also demonstrated improved sGAG secretion and chondrogenic and integrin gene expression with the increased negative charge in vitro. Similar results were also observed in subcutaneous implantation in vivo, where higher secretions of sGAG, SOX9 and collagen type II proteins were found in the collagen hydrogels with higher negative charge. Together, our results demonstrated that more negative charges introduced into the collagen hydrogel microenvironment would enhance the chondrogenic differentiation of BMSCs in vitro and in vivo. This revealed that the electrical properties are an important consideration in designing future collagen hydrogels for cartilage regeneration.

Injectable hydrogels attract a great deal of attention due to their in situ formation in vivo, which minimizes the risk of major trauma during the surgical procedure as compared to traditional hydrogel scaffolds. Click chemistry is widely used in the preparation of injectable hydrogels, although some of them require photoinitiators or catalysts that may be cytotoxic and impair the proliferation of encapsulated cells. In the present study, an injectable dual crosslinking hydrogel was designed by a thiol–ene click reaction and conversion between sulfhydryl and disulfide bonds at a neutral pH. The sulfhydryl group and acrylate group were successfully grafted onto HA and characterized using FT-IR and 1H-NMR. The gelation time, morphology, injection force, swelling, degradation, mechanical properties and drug release behavior of the hydrogel varied with the molecular weight of the hydrogel (Mw = 0.1, 1.0, 2.0 MDa). MTT, FDA/PI staining and scanning electron microscopy (SEM) showed that the encapsulated L929 proliferated well in different molecular weight hydrogels. It was also observed that in a low molecular weight (0.1 MDa) hydrogel system, the cells were better distributed and secreted more extracellular matrix. Subcutaneous injection in mice also demonstrated that hydrogels could support the proliferation of encapsulated L929 cells in vivo, and no significant infiltration of peripheral cells into the interior of the material was observed. These results indicate that this injectable hyaluronan-based hydrogel is an excellent cell protectant and exhibits good biocompatibility, with potential for biomedical applications such as cell delivery and postoperative anti-adhesion.

The surface structure of calcium phosphate (CaP) ceramic plays an important role in its osteoinductivity; however, little is known about its effects on osteoclastogenesis. In this study, an intramuscular implantation model suggested a potential relationship between hydroxyapatite (HA)-induced bone formation and osteoclast appearance in the non-osseous site, which might be modulated by scaffold surface structure. Then, three dense HA discs with different grain sizes from biomimetic nanoscale (∼100 nm) to submicron scale (∼500 nm) were fabricated via distinct sintering procedures, and their impacts on osteoclastic differentiation of RAW 264.7 macrophages under RANKL stimulation were further investigated. Our results showed that compared with the ones in the submicron-scale dimension, nano-structured HA discs markedly impaired osteoclastic formation and function, as evidenced by inhibited cell fusion, reduced osteoclast size, less-defined actin ring, increased osteoclast apoptosis, suppressed expression of osteoclast specific genes and proteins, decreased TRAP-positive cells, and hampered resorption activity. This demonstrated that the surface structure of CaP ceramics has a great influence on osteoclastogenesis, which might be further related to its osteoinductive capacity. These findings might not only help us gain insight into biomolecular events during CaP-involved osteoinduction, but also offer a principle for designing orthopaedic implants with an ability of regulating both osteogenesis and osteoclastogenesis to achieve the desired performance.

Extracellular matrix (ECM) scaffolds made from decellularized natural cartilage have been successfully used in cartilage lesion repair, but allogeneic cartilage donors are always in shortage and xenogeneic cartilage tissues may have the risk of unknown disease transfer. In this study, we constructed artificial bionic cartilage microspheres by encapsulating MSCs in collagen microspheres and cultured in a chondrogenic-inducing medium. Then, acellular matrix microsphere (BCAMM) scaffolds were fabricated from the cultured microspheres at three different developmental stages. A novel technique was introduced to fabricate BCAMM scaffolds, which enabled the production and utilization of the scaffolds in a short time. Due to the differences in surface morphologies and biological compositions, the three BCAMM scaffolds showed different chondrogenic effects. The 10-day BCAMM (10-BCAMM) scaffold showed the best overall results, successfully inducing MSC chondrogenesis without any additional fetal bovine serum or induction components (TGF-β or dexamethasone). In comparison, the 5-day BCAMM (5-BCAMM) scaffold showed potential osteogenic effects. The advantages of micron-sized BCAMMs are outlined, specifically in the easier decellularization process without grinding, homogeneous cell seeding and infiltration, chondrogenic induction and better fitting to the irregular lesion shape.

Osteochondral defects occur in the superficial cartilage region, intermediate calcified cartilage, and subchondral bone. Due to the limited regenerative capacity and complex zonal structure, it is critically difficult to develop strategies for osteochondral defect repair that could meet clinical requirements. In this study, type I collagen (Col I) and BCP ceramics were used to fabricate a new bi-layer scaffold for regeneration in osteochondral defects. The in vitro studies showed that the bi-layer scaffold provided special functions for cell migration, proliferation and secretion due to the layered scaffold structure. The in vivo results demonstrated that the bi-layered scaffold could effectively promote the regeneration of both the cartilage and the subchondral bone, and the newly formed cartilage layer, with a similar structure and thickness to the natural cartilaginous layer, could seamlessly integrate with the surrounding natural cartilage and regenerate an interface layer to mimic the native osteochondral structure.

Small interference RNA (siRNA) has demonstrated unprecedented potential as a therapy for drug-resistant cancer. However, efficient cellular delivery is still a challenge due to hydrolytic sensitivity and poor cellular uptake of siRNA. Strategies to conjugate siRNA to the delivery vehicle and activate innate immunity have shown low in vivo efficacy. Therefore, nanomedicine approaches have become the main focus in this field. B-cell lymphoma 2 (Bcl-2) is the founding member of the Bcl-2 family of regulatory proteins that regulate cell death (apoptosis), by either inducing (pro-apoptotic) or inhibiting (anti-apoptotic) apoptosis. In this report, a nanomedicine system is constructed using Bcl-2 siRNA as the therapeutic agent and mesoporous polymer nanosphere (MPN) carriers to both improve cellular internalization and achieve Bcl-2 silencing and cell apoptosis. MPNs were prepared through a two-stage hydrothermal process at two different temperatures, which was deliberately designed to form nanospheres via self-assembly and create mesoporous structures by removing the pore-forming templates. Such MPNs were proved to be biodegradable. Without any carbonization process, MPNs still keep many active groups which endow them with excellent properties for functionalization purposes. Finally, the FA-targeted-Bcl-2-siRNA-loaded nanoparticles were constructed by a layer-by-layer assembly by electrostatic interactions after nitrification. These nanoparticles were efficiently delivered into breast cancer (BC) cells, showing a significant sequence-specific inhibition of Bcl-2 mRNA expression in BC cells, enhanced tumor cell apoptosis and tumor therapeutic efficacy. Taken together, this study establishes a novel therapeutic system for cancer therapy.

In this work, spindle-shaped cells derived from bone marrow were separated from the regenerated tissue of an osteochondral defect in a SD rat model. These cells were defined as MSC-like cells because they revealed similar features with MSCs, as demonstrated by the cell morphological observation, H&E staining of the regenerated tissue, typical MSC surface markers, and the differentiation potential assays in osteogenesis, adipogenesis, and chondrogenesis. Bioinert agarose and bioactive collagen I hydrogels were filled in the osteochondral defects to investigate the effects of different biomaterials on the MSC-like cell migration, differentiation, and osteochondral regeneration. After one month's surgery, the defect site was evaluated by histologic staining, IHC staining and micro-CT. These results indicated that the bioinert agarose significantly inhibited cell migration and tissue regeneration, while the bioactive collagen I hydrogel was found to have a therapeutic effect. It would be tempting to speculate that the specifically designed scaffolds that facilitated cell migration were more propitious to osteochondral defect healing. The migration of the MSC-like cells into the osteochondral defect had produced promising outcomes and showed therapeutic potency. In the future, additional work will focus on elucidating the sophisticated mechanisms underlying the origin of the MSC-like cells, function and consequences of cell migration to understand the treatment of osteochondral defects.

Development of an in situ injectable hydrogel with controlled-release of bioactive molecules is an effective strategy to maintain chondrocyte phenotype and promote cartilage formation for clinical applications. In this study, we developed an injectable thiolated icariin functionalized hyaluronic acid/collagen hydrogel (HIC) under physiological conditions. In order to covalently introduce icariin (Ica) into an injectable hydrogel and increase its loading, thiolated icariin (Ica-SH) was successfully synthesized. Ica-SH could not only decrease the cytotoxicity of Ica, but also increase the Ica loading in hydrogels. Compared to hyaluronic acid/collagen hydrogels, HICs facilitated chondrocyte proliferation, maintained chondrocyte phenotype and promoted the secretion of the cartilage extracellular matrix. Overall, this study suggests that HIC has great potential to be developed as an effective and less invasive candidate for clinical application in articular cartilage repair.

Stimuli-responsive nanotechnology-mediated drug co-delivery system is a notable strategy to improve access of the systemically administered chemotherapeutics to the tumors. Herein, a tailor-made 2,3-dimethylmaleic-anhydride-poly(ethylene glycol)-e-poly-l-lysine-doxorubicin /lapatinib polymeric nanoplatform (DMMA-P-DOX/LAP) for synergistically eliminating breast cancer is developed by encapsulating lapatinib into dual-pH responsive charge switchable biopolymer-doxorubicin conjugate nanoparticles. The physicochemical properties of polymeric nanoparticles are conducive to their stable circulation in the physiological condition, but reverse the surface charge from negative to positive ultrasensitively in slightly acidic tumor microenvironment, facilitating cell internalization and deep tumor penetration. Subsequently, DOX and LAP are synchronously released into the cytoplasm in response to the significantly increased acidity of intracellular environment. As a result, the combination therapy by DMMA-P-DOX/LAP nanoparticles compels the solid tumors to contract significantly or even vanish completely in the MCF-7 tumor model, moreover, the structural composition with amino acid and bioinert PEG ensures the favorable biosecurity of the co-delivery system in vivo. This dual-pH responsive nanotechnology-mediated drug co-delivery system provides great potentials for safe and effective cancer therapy.

Hydrogels hold great promise for tissue engineering and as cell delivery vehicles, but their application is greatly limited by the mass depletion of cells after encapsulation due to their low cell affinity and high cell constraints. In this study, using human osteoblast-like cells (MG63) as a model for anchorage-dependent cells (ADCs), alginate hydrogels incorporated with collagen microcarriers (MCs) and collagenase (GCE) were constructed to promote the cellular functional establishment. In this system, the cell-laden microcarriers as cellular 'anchors' provided a cell-affinitive interface between cells and hydrogels, while the collagenase promoted the degradation of collagen microspheres and thus provided adequate room for cell spreading and migration. Compared to the pure alginate hydrogel (Gel), the alginate hydrogel/microcarrier composite (GC) improved initial cell spreading by supplying a cell adhesive interface, while GCE with both microcarriers and collagenase exhibited much better cell spreading, proliferation and differentiation, which is attributed to the sustainable supply of both a cell adhesive interface and cell spreading room. The composites showed potential as an injectable vehicle for the conveyance of ADCs and tissue engineering.

Surface tension driven assembly is a simple and rapid strategy in “modular” tissue engineering to fabricate three-dimensional (3D) structures with predefined geometrical and biological features. Besides the biocompatibility for encapsulated cells, the mechanical and hydrophilic properties of the microgel are the utmost two factors for assembly and structure preservation. Herein, we developed a new composite hydrogel based on photocrosslinkable methacrylated gelatin (GelMA) and methacrylated alginate (AlgMA) for modular tissue engineering assembly. Based on the high cellular bioactivity of GelMA, inflexible AlgMA with a large number of hydrophilic groups was introduced to optimize the hydrogel assembly performance. The results revealed that the mechanical stiffness, swelling ratio and hydrophilicity were improved greatly, and the nutrient permeability as well as the cellular activity remained at a relatively high level simultaneously. The enhanced stiffness and hydrophilicity were helpful for the surface tension driven assembly, and the high nutrient permeability was conducive to the activity of encapsulated cells. Moreover, microgels with different shapes were fabricated, and microengineered complex structures constructed with spatial organized cell distribution and specific functions, such as osteon-like structure containing both osteogenic and vascularized area, were successfully generated by “Lock and key” and “Concentric double-ring” assemblies. The composite hydrogel provides a potential candidate material for modular tissue engineering.