Thermochemotherapy Meets Tissue Engineering for Rheumatoid Arthritis Treatment

Rheumatoid arthritis (RA) is an autoimmune disease that progresses from inflammation to cartilage destruction. Inspired by the similar characteristics of inflammatory granulation tissue to those of tumors, the newly emerged tumor therapy called thermochemotherapy is proposed to treat RA. Meanwhile, the repair of cartilage injury via tissue engineering is paid attention simultaneously. A first-line antirheumatic drug (MTX; methotrexate) and transforming growth factor β1 (TGF-β1) are loaded in nano-Fe₃O₄ composite chitosan-polyolefin to construct a multifunctional hydrogel (DN-Fe-MTX-TGFβ1). The mechanical properties of the hydrogel are equivalent to that of articular cartilage to guarantee its role as a scaffold. A long-term release ability and the magnetocaloric properties of the hydrogel assure its effect to provide sustained local thermochemotherapy. The effective ability of the hydrogel for both anti-inflammation and cartilage repair is demonstrated. This work indicates a promising way to combine thermochemotherapy and tissue engineering for the effective treatment of RA for the first time.     

Regulation of the Stem Cell–Host Immune System Interplay Using Hydrogel Coencapsulation System with an Anti‐Inflammatory Drug

The host immune system is known to influence mesenchymal stem cell (MSC)‐mediated bone tissue regeneration. However, the therapeutic capacity of hydrogel biomaterial to modulate the interplay between MSCs and T‐lymphocytes is unknown. Here it is shown that encapsulating hydrogel affects this interplay when used to encapsulate MSCs for implantation by hindering the penetration of pro‐inflammatory cells and/or cytokines, leading to improved viability of the encapsulated MSCs. This combats the effects of the host pro‐inflammatory T‐lymphocyte‐induced nuclear factor kappaB pathway, which can reduce MSC viability through the CASPASE‐3 and CASPASE‐8 associated proapoptotic cascade, resulting in the apoptosis of MSCs. To corroborate rescue of engrafted MSCs from the insult of the host immune system, the incorporation of the anti‐inflammatory drug indomethacin into the encapsulating alginate hydrogel further regulates the local microenvironment and prevents pro‐inflammatory cytokine‐induced apoptosis. These findings suggest that the encapsulating hydrogel can regulate the MSC‐host immune cell interplay and direct the fate of the implanted MSCs, leading to enhanced tissue regeneration.     

Smart Biomaterials for Articular Cartilage Repair and Regeneration

Articular cartilage defects bring about disability and worldwide socioeconomic loss, therefore, articular cartilage repair and regeneration is recognized as a global issue. However, due to its avascular and nearly acellular characteristic, cartilage tissue regeneration ability is limited to some extent. Despite the availability of various treatment methods, including palliative drugs and surgical regenerative therapy, articular cartilage repair and regeneration still face major challenges due to the lack of appropriate methods and materials. Smart biomaterials can regulate cell behavior and provide excellent tissue repair and regeneration microenvironment, thus inducing articular cartilage repair and regeneration. This process is adjusted by controlling drug/bioactive factors release via responding to exogenous/endogenous stimuli, tailoring materials’ structure and function similar to native cartilage or providing physiochemical and physical signaling factors. Herein, smart biomaterials, recently applied in articular cartilage repair and regeneration, are elaborated from two aspects: smart drug release system and smart scaffolds. Furthermore, articular cartilage and its defects and advanced manufacturing techniques of smart biomaterials are discussed in brief. Finally, perspectives for smart biomaterials used in articular cartilage repair and regeneration are presented and the clinical translation of smart biomaterials is emphasized.     

Creating a Living Hyaline Cartilage Graft Free from Non‐Cartilaginous Constituents: An Intermediate Role of a Biomaterial Scaffold

A novel living hyaline cartilage graft (LhCG) with controllable dimensions and free of non‐cartilaginous constituents for articular regeneration is developed. As a living graft for regenerative medicine, LhCG is purely living tissue based and truly scaffold‐free. The process of neotissue formation in LhCG is mediated by an interim biomaterial‐based novel scaffolding system. This design highlights a philosophy of using biomaterials in engineered regenerative medicine as a transient guiding facility rather than a permanent part of substitute. The fabrication is designed and practiced in a continuous and integrated process, which attributes to its simplicity in operation. Because of the intrinsic non‐cell‐adhesive property of hydrogel scaffolds, articular chondrocytes’ phenotype is always preserved throughout the whole procedure, which has been tested and approved both in vitro and in vivo. In situ grafting trials in a rabbit model showcase high success rates in both cartilage repair and graft‐host integration. Beyond cartilage repair, this LhCG model may provide a living‐tissue‐based open platform or niche for multi‐tissue regenerations.     

Recent Advances in Design of Functional Biocompatible Hydrogels for Bone Tissue Engineering

Bone related diseases have caused serious threats to human health owing to their complexity and specificity. Fortunately, owing to the unique 3D network structure with high aqueous content and functional properties, emerging hydrogels are regarded as one of the most promising candidates for bone tissue engineering, such as repairing cartilage injury, skull defect, and arthritis. Herein, various design strategies and synthesis methods (e.g., 3D-printing technology and nanoparticle composite strategy) are introduced to prepare implanted hydrogel scaffolds with tunable mechanical strength, favorable biocompatibility, and excellent bioactivity for applying in bone regeneration. Injectable hydrogels based on biocompatible materials (e.g., collagen, hyaluronic acid, chitosan, polyethylene glycol, etc.) possess many advantages in minimally invasive surgery, including adjustable physicochemical properties, filling irregular shapes of defect sites, and on-demand release drugs or growth factors in response to different stimuli (e.g., pH, temperature, redox, enzyme, light, magnetic, etc.). In addition, drug delivery systems based on micro/nanogels are discussed, and its numerous promising designs used in the application of bone diseases (e.g., rheumatoid arthritis, osteoarthritis, cartilage defect) are also briefed in this review. Particularly, several key factors of hydrogel scaffolds (e.g., mechanical property, pore size, and release behavior of active factors) that can induce bone tissue regeneration are also summarized in this review. It is anticipated that advanced approaches and innovative ideas of bioactive hydrogels will be exploited in the clinical field and increase the life quality of patients with the bone injury.     

3D Printed Cartilage‐Like Tissue Constructs with Spatially Controlled Mechanical Properties

Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage‐like tissue is fabricated using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a bath composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel (MPa order compressive modulus) is developed as an extracellular matrix (ECM) with self‐healing properties. Within this bath supplemented with thrombin, human mesenchymal stem cell (hMSC) spheroids embedded in fibrinogen are 3D bioprinted, creating a soft microenvironment composed of fibrin (kPa order compressive modulus) that simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids present high viability and chondrogenic‐like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to locally bioprint a soft and cell stimulating biomaterial inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro‐ and macromechanical properties of the 3D printed tissues such as cartilage.     

Glycopeptide-Based Multifunctional Hydrogels Promote Diabetic Wound Healing through pH Regulation of Microenvironment

Excessive inflammation, bacterial infection, and blocked angiogenesis make diabetic wound healing challenging. Multifunctional wound dressings have several advantages in diabetic wound healing. In addition, the pH regulation of the microenvironment is shown to be a key factor that promotes skin regeneration through cellular immune regulation. However, few reports have focused on the development of functional dressings with the ability to regulate the pH microenvironment and promote diabetic wound healing. This study presents a novel approach for regulating the pH microenvironment of diabetic wound sites using a glycopeptide-based hydrogel consisting of modified hyaluronic acid and poly(6-aminocaproic acid). This hydrogel forms a network through Schiff base interactions and metal complexation, which suppresses inflammation and accelerates angiogenesis during wound healing. Hydrogels not only have adequate mechanical properties and self-healing ability but can also support tissue adhesion. They can also promote the secretion of inducible cAMP early repressor, which promotes the polarization of macrophages toward the M2 type. The in vivo results confirm that hydrogel promotes diabetic wound repair and skin regeneration by exerting rapid anti-inflammatory effects and promoting angiogenesis. Therefore, this hydrogel system represents an effective strategy for treating diabetic wounds.     

3D Molecularly Functionalized Cell‐Free Biomimetic Scaffolds for Osteochondral Regeneration

Clinically, cartilage damage is frequently accompanied with subchondral bone injuries caused by disease or trauma. However, the construction of biomimetic scaffolds to support both cartilage and subchondral bone regeneration remains a great challenge. Herein, a novel strategy is adopted to realize the simultaneous repair of osteochondral defects by employing a self‐assembling peptide hydrogel (SAPH) FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine) to coat onto 3D‐printed polycaprolactone (PCL) scaffolds. Results show that the SAPH‐coated PCL scaffolds exhibit highly improved hydrophilicity and biomimetic extracellular matrix (ECM) structures compared to PCL scaffolds. In vitro experiments demonstrate that the SAPH‐coated PCL scaffolds promote the proliferation and osteogenic differentiation of rabbit bone mesenchymal stem cells (rBMSCs) and maintain the chondrocyte phenotypes. Furthermore, 3% SAPH‐coated PCL scaffolds significantly induce simultaneous regeneration of cartilage and subchondral bone after 8‐ and 12‐week implantation in vivo, respectively. Mechanistically, by virtue of the enhanced deposition of ECM in SAPH‐coated PCL scaffolds, SAPH with increased stiffness facilitates and remodels the microenvironment around osteochondral defects, which may favor simultaneous dual tissue regeneration. These findings indicate that the 3% SAPH provides efficient and reliable modification on PCL scaffolds and SAPH‐coated PCL scaffolds appear to be a promising biomaterial for osteochondral defect repair.     

Cell Development Enhanced Bionic Silk Hydrogel on Remodeling Immune Pathogenesis of Spinal Cord Injury via M2 Polarization of Microglial

Due to the complex spatial-temporal pathophysiology of spinal cord injury (SCI), effective modulation of SCI-specific inflammatory pathogenesis to achieve desirable therapeutic effects on functional recovery still remains challenging. Herein, cell-enhanced photocrosslinked silk fibroin hydrogels with extracellular matrix-mimicking cues of mechanical properties and RGD (Arg-Gly-Asp) signals are gelled in situ to fill the lesion site to modulate injury-induced neuroinflammation and promote neurite regrowth after SCI. The bionic hydrogel system provides biomimetic mechanical cues to promote neuronal differentiation of neural stem/progenitor cells (NPCs) and neurite growth by activating YAP nuclear expression. Importantly, favored by the strong capacity of silk fibroin hydrogels on macrophage/microglia recruitment, NPCs encapsulated hydrogel (NPCs@SFRGD0.1) effectively promotes recruited macrophages/microglia to M2 polarization in the lesion site by releasing S100A4 and thereby remodels the inflammatory microenvironment after SCI. Moreover, NPCs@SFRGD0.1 successfully reduces glial scar formation and accelerates corticospinal tract axon regrowth to improve locomotor recovery. Overall, this work contributes to illustrating the therapeutic mechanism of NPCs development based biomaterial therapies on modulating inflammatory microenvironment and this NPCs enhanced silk fibroin hydrogel provides a promising therapeutic strategy for SCI.     

A Macroporous Hydrogel Dressing with Enhanced Antibacterial and Anti‐Inflammatory Capabilities for Accelerated Wound Healing

Here, a novel macroporous hydrogel dressing is presented that can accelerate wound healing and guard against bacteria‐associated wound infection. Carboxymethyl agarose (CMA) is successfully prepared from agarose. The CMA molecular chains are cross‐linked by hydrogen bonding to form a supramolecular hydrogel, and the hydroxy groups in the CMA molecules complex with Ag⁺ to promote hydrogel formation. This hydrogel composite exhibits pH‐responsiveness and temperature‐responsiveness and releases Ag⁺, an antibacterial agent, over a prolonged period of time. Moreover, this hydrogel exhibits outstanding cytocompatibility and hemocompatibility. In vitro and in vivo investigations demonstrate that the hydrogel has enhanced antibacterial and anti‐inflammatory capabilities and can significantly accelerate skin tissue regeneration and wound closure. Astonishingly, the hydrogel can cause the inflammation process to occur earlier and for a shorter amount of time than in a normal process. Given its excellent antibacterial, anti‐inflammatory, and physicochemical properties, the broad application of this hydrogel in bacteria‐associated wound management is anticipated.     

A Single Component Conducting Polymer Hydrogel as a Scaffold for Tissue Engineering

Conducting polymers (CPs) have exciting potential as scaffolds for tissue engineering, typically applied in regenerative medicine applications. In particular, the electrical properties of CPs has been shown to enhance nerve and muscle cell growth and regeneration. Hydrogels are particularly suitable candidates as scaffolds for tissue engineering because of their hydrated nature, their biocompatibility, and their tissue‐like mechanical properties. This study reports the development of the first single component CP hydrogel that is shown to combine both electro‐properties and hydrogel characteristics. Poly(3‐thiopheneacetic acid) hydrogels were fabricated by covalently crosslinking the polymer with 1,1′‐carbonyldiimidazole (CDI). Their swelling behavior was assessed and shown to display remarkable swelling capabilities (swelling ratios up to 850%). The mechanical properties of the networks were characterized as a function of the crosslinking density and were found to be comparable to those of muscle tissue. Hydrogels were found to be electroactive and conductive at physiological pH. Fibroblast and myoblast cells cultured on the hydrogel substrates were shown to adhere and proliferate. This is the first time that the potential of a single component CP hydrogel has been demonstrated for cell growth, opening the way for the development of new tissue engineering scaffolds.     

Magnesium Gradient-Based Hierarchical Scaffold for Dual-Lineage Regeneration of Osteochondral Defect

Osteochondral regeneration remains a great challenge due to the limited self-healing ability and the complexity of its hierarchical structure and composition. Mg²⁺ and hypoxia are two effective modulators in boosting chondrogenesis. To this end, a double-layered scaffold (D) consisting of a hydrogel layer on a porous cryogel is fabricated to mimic the hierarchical structure of osteochondral tissue. An Mg²⁺ gradient is incorporated into the double-layered scaffold with hypoxia-mimicking deferoxamine (DFO) embedded in the hydrogel (D-Mg-DFO), which remarkably augments the dual-lineage regeneration of both cartilage and subchondral bone. The higher Mg²⁺ supplementation from the upper hydrogel, associated with its hypoxia-mimicking situation and small pore size, exhibits promotive effects on chondrogenic differentiation. The lower Mg²⁺ supplementation from the bottom cryogel, associated with its interconnected macroporous structure, achieves multiple contributions in stem cell migration from bone marrow cavity, matrix mineralization, and osteogenesis. Furthermore, rabbits’ trochlea osteochondral defects are established to evaluate the regenerative outcome. Compared to control scaffolds containing only Mg²⁺ or DFO, the D-Mg-DFO scaffold presents the best regenerative effect under the synergistic contribution of multiple factors. Overall, this work provides a new design of scaffold toward an effective repair of cartilage defect.     

Mechanically Activated Microcapsules for “On‐Demand” Drug Delivery in Dynamically Loaded Musculoskeletal Tissues

Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimuli‐responsive delivery systems can facilitate “on‐demand” release of therapeutics in response to a variety of physiologic triggering mechanisms (e.g., pH, temperature). However, few systems to date have taken advantage of mechanical inputs from the microenvironment to initiate drug release. Here, mechanically activated microcapsules (MAMCs) are designed to deliver therapeutics in response to the mechanically loaded environment of regenerating musculoskeletal tissues, with the ultimate goal of furthering tissue repair. To establish a suite of microcapsules with different thresholds for mechanoactivation, MAMC physical dimensions and composition are first manipulated, and their mechano‐response under both direct 2D compression and in 3D matrices mimicking the extracellular matrix properties and dynamic loading environment of regenerating tissue, is evaluated. To demonstrate the feasibility of this delivery system, an engineered cartilage model is used to test the efficacy of mechanically instigated release of transforming growth factor‐β3 on the chondrogenesis of mesenchymal stem cells. These data establish a novel platform by which to tune the release of therapeutics and/or regenerative factors based on the physiologic mechanical loading environment and will find widespread application in the repair and regeneration of musculoskeletal tissues.     

Rational Design of Bioactive Hydrogels toward Periodontal Delivery: From Pathophysiology to Therapeutic Applications

Periodontitis is a biofilm-induced, host-mediated inflammatory disease that results in periodontal tissue destruction. The design of functional biomaterials based on disease pathophysiology is essential for enhancing their therapeutic effects in periodontitis treatment. As promising localized drug delivery systems and tissue engineering scaffolds, hydrogels have gained significant interest for controlled and sustained release of bioactive agents in periodontal applications. The rational design of bioactive hydrogels can facilitate bacterial control and modulate destructive host inflammation, thereby preventing the progression of periodontitis. In this review, the pathophysiological mechanisms underlying periodontitis as fundamental principles for the design of functional hydrogel systems are first introduced. In the following part, an overview is systematically provided of the types and functions of the bioactive hydrogel systems loaded with anti-bacterial and anti-inflammatory agents for periodontal delivery. Finally, the remaining challenges and future perspectives of hydrogel delivery systems for periodontal applications are proposed. It is believed that this review will inspire the rational design and development of innovative functional hydrogel biomaterials toward periodontal therapy.     

Multifunctional DNA Hydrogels with Hydrocolloid-Cotton Structure for Regeneration of Diabetic Infectious Wounds

Currently, diabetic infectious wound treatments remain a significant challenge for regenerative medicine due to the unicity of clinical dressings, which lack systemic multifunctional wound dressings with high absorbability, customizable shape, rapid self-healing, guiding tissue regeneration, and restoring physiological functions. Here, a multifunctional DNA hydrogel is conveniently obtained through grafting DNA units and polyethyleneimine dynamic cross-linking and doped heating function black phosphorus quantum dots. The obtained DNA hydrogel features excellent exudate absorption performance, adjustable heating ability, mechanical behavior, self-healing ability, writability, tissue adhesion, and antibacterial properties. The incorporation of procyanidin B2 (OPC B2) endows the DNA hydrogels with renowned scavenging free radicals and antioxidant properties. Furthermore, the DNA hydrogel dressing can promote the transformation of macrophages from pro-inflammatory M1 into repairing M2 phenotype, keeping the wound in a stable remodeled state. Astonishingly, the DNA hydrogel dressing can activate neurons to transform into a repair state, accelerating skin nerve regeneration and angiogenesis. Beyond that, it can recruit myeloid cells to activate the adaptive immune response, enhancing the ability of DNA hydrogel dressing to promote tissue regeneration, thereby promoting hair follicle and hair regeneration. Therefore, this advanced collaborative strategy provides an effective method for cascade management of clinical guided tissue regeneration.     

Self-Amplification Immunomodulatory Strategy for Tissue Regeneration in Diabetes Based on Cytokine-ZIFs System

Dysfunctional macrophages and excessive inflammatory responses lead to severe tissue regeneration disorders in diabetes. Herein, a function-oriented self-amplification immunomodulatory (SAI) strategy based on an interleukin-33 (IL-33) loaded zeolitic imidazolate frameworks (IL@ZIF) nano-platform is proposed to treat tissue regeneration disorders by restoring macrophage function and reconstructing immune microenvironment in diabetes. It is found that ZIFs effectively protect IL-33 from premature degradation. In the wound area, the released Zn²⁺ not only improves the antioxidant capacity of macrophages to avoid reactive oxygen species-induced dysfunction, but also upregulates IL-33 receptor (ST2L) expression and triggers M2 macrophages polarization. Subsequently, the released IL-33 significantly amplifies M2 macrophage polarization through IL-33/ST2L signaling, resulting in a reversal of the pro-inflammatory microenvironment of diabetic wounds. This synergistic effect endows the nano-platform with an excellent ability to accelerate tissue regeneration in vitro and in vivo. Overall, this IL@ZIF mediated function-oriented SAI strategy provides new alternatives for the treatment of tissue regeneration disorders in diabetes.     

Injection of ROS-Responsive Hydrogel Loaded with Basic Fibroblast Growth Factor into the Pericardial Cavity for Heart Repair

Myocardial infarction, among other ischemic heart diseases, is the major cause of mortality and morbidity for patients who have heart diseases. Timely reperfusion of the ischemic myocardium is the most effective way to treat myocardial infarction. However, blood reperfusion to the ischemic tissues leads to an overproduction of toxic reactive oxygen species (ROS), which can further exacerbate myocardial damage on top of ischemic injury. ROS has been used as a diagnostic marker and therapeutic target for ischemia-reperfusion (I/R) injury and as an environmental stimulus to trigger drug release. In this study, a ROS-sensitive cross-linked poly(vinyl alcohol) (PVA) hydrogel is synthesized to deliver basic fibroblast growth factor (bFGF) for myocardial repair. The therapeutic gel is injected into the pericardial cavity. Upon delivery, the hydrogel spread on the surface of the heart and form an epicardiac patch in situ. In a rat model of I/R injury, bFGF released from the gel could penetrate the myocardium. Such intervention protects cardiac function and reduces fibrosis in the post-I/R heart, with enhanced angiomyogenesis. Furthermore, the safety and feasibility of minimally invasive injection and access into the pericardial cavity in both pigs and human patients are demonstrated.     

Injectable Polypeptide‐Protein Hydrogels for Promoting Infected Wound Healing

Protein is the key composition of all tissues, which has also been widely used in tissue engineering due to its superior biocompatibility and low immunogenicity. However, natural protein usually lacks active functions such as vascularization, osteo‐induction, and neural differentiation, which limits its further applications as a functional biomaterial. Here, based on the mimetic extracellular matrix feature of bovine serum albumin, injectable polypeptide‐protein hydrogels with vascularization and antibacterial abilities are constructed successfully via coordinative cross‐linking of sulfydryl groups with silver ions (Ag+) for the regeneration of infected wound. In this protein hydrogel system, (Ag+), acting as crosslinkers, can not only provide a sterile microenvironment and a strong and robust antibacterial ability but also introduce K₂(SL)₆K₂ (KK) polypeptide, which endows the hydrogel with vascularization behavior. Furthermore, the in vivo data show that the polypeptide‐protein hydrogel has a considerable collagen deposition and vascularization abilities in the early stage of wound healing, resulting in rapid new tissue regeneration featured with newly appeared hair follicles. Altogether, this newly developed multifunctional 3D polypeptide‐protein hydrogel with vascularization, antibacterial properties, and hair follicle promotion can be a promising approach in biomedical fields such as infected wound healing.     

Injectable 3D-Printed Porous Scaffolds for Adipose Stem Cell Delivery and Endometrial Regeneration

Stem-cell-based therapeutic strategies are promising in the clinical treatment of intrauterine adhesions (IUAs), while endometrial regeneration still hardly restores the structure and function of the endometrium because of the inadequate microenvironment for the grafted stem cells and subsequent limited therapeutic efficiency. Herein, an injectable porous hydrogel scaffold (PH scaffold) with customizable shapes is presented by using a microfluidic-based 3D printing technique for adipose-derived stem cells (ADSCs) delivery to enhance endometrial regeneration. These scaffolds display a controllable interconnected porous structure, which not only facilitates the encapsulation of ADSCs within the scaffold but also supports the recovery to their original shapes after injection. Furthermore, the cell viability of the laden ADSCs is well-maintained post-injection, exhibiting promotive effects on cell migration, proliferation, and tube formation. Based on these features, an ADSCs-laden PH scaffold with a hollow endometrium-mimicking morphology is designed and in situ injected into the damaged endometrium in rats of IUAs. These results show that the ADSCs-laden PH scaffolds can enhance functional endometrial regeneration by suppressing the inflammatory response, promoting cell proliferation, and improving vascularization. Thus, it is believed that such unique 3D-printed porous scaffolds are promising candidates for cell delivery, which also provides a minimally-invasive and effective strategy for endometrial regeneration.     

3D Printing Hydrogel Scaffolds with Nanohydroxyapatite Gradient to Effectively Repair Osteochondral Defects in Rats

Osteochondral (OC) defects pose an enormous challenge with no entirely satisfactory repair strategy to date. Herein, a 3D printed gradient hydrogel scaffold with a similar structure to that of OC tissue is designed, involving a pure hydrogel-based top cartilage layer, an intermediate layer for calcified cartilage with 40% (w w⁻¹) nanohydroxyapatite (nHA) and 60% (w w⁻¹) hydrogel, and a 70/30% (w w⁻¹) nHA/hydrogel-based bottom subchondral bone layer. This study is conducted to evaluate the efficacy of the scaffold with nHA gradients in terms of its ability to promote OC defect repair. The fabricated composites are evaluated for physicochemical, mechanical, and biological properties, and then implanted into the OC defects in 56 rats. Overall, bone marrow stromal cells (BMSCs)-loaded gradient scaffolds exhibit superior repair results as compared to other scaffolds based on gross examination, micro-computed tomography (micro-CT), as well as histologic and immunohistochemical analyses, confirming the ability of this novel OC graft to facilitate simultaneous regeneration of cartilage-subchondral bone.