Materials Suitable for Osteochondral Regeneration

Osteochondral defects affect articular cartilage, calcified cartilage, and subchondral bone. The main problem that they cause is a different behavior of cell tissue in the osteochondral and bone part. Articular cartilage is composed mainly of collagen II, glycosaminoglycan (GAG), and water, and has a low healing ability due to a lack of vascularization. However, bone tissue is composed of collagen I, proteoglycans, and inorganic composites such as hydroxyapatite. Due to the discrepancy between the characters of these two parts, it is difficult to find materials that will meet all the structural and other requirements for effective regeneration. When designing a scaffold for an osteochondral defect, a variety of materials are available, e.g., polymers (synthetic and natural), inorganic particles, and extracellular matrix (ECM) components. All of them require the accurate characterization of the prepared materials and a number of in vitro and in vivo tests before they are applied to patients. Taken in concert, the final material needs to mimic the structural, morphological, chemical, and cellular demands of the native tissue. In this review, we present an overview of the structure and composition of the osteochondral part, especially synthetic materials with additives appropriate for healing osteochondral defects. Finally, we summarize in vitro and in vivo methods suitable for evaluating materials for restoring osteochondral defects.


INTRODUCTION
The osteochondral unit is a complex tissue region that transitions from a top layer of hyaline (articular) cartilage, through calcified cartilage into the subchondral bone layer.This tissue is found in areas of the body critical for locomotion. 1 Osteochondral defects involve cartilage and subchondral bone and cancellous bone beneath the cartilage that form the osteochondral unit.They can be caused by injury or diseases, and are difficult to heal due to the lack of vasculature in the articular cartilage. 1,2These defects can occur as a partial defect where there is only injury to the cartilage layer (mostly composed of collagen fibers and GAGs), or a full-thickness defect which also includes the subchondral bone layer (mostly composed of calcium phosphate and hydroxyapatite). 1 The mineral content increases from cartilage to bone, while the collagen and water concentration diminish. 3The mechanical properties of bone differ based on the load orientation (anisotropy) and the speed at which the load is applied (viscoelasticity). 4Structurally, pore size, porosity, and vascularization increase from cartilage to bone Figure 1.Mechanically, compressive modulus increases from cartilage to osseous tissue. 5any studies are focused on strategies for healing the subchondral unit.Due to the complicated architecture of this tissue, biphasic and multiphasic scaffolds containing two or more different layers could mimic the physiology and function of this tissue with a higher degree of similarity. 6,7The new materials have to reflect the native osteochondral tissue that can be a multilayer system (bilayer, trilayer) using 3D printed technologies. 8−10 A good restorative effect can be achieved if it has a hierarchical structure, optimal porosity and mechanical properties, and contains bioactive components.A multilayer system should simultaneously meet the following requirements: (i) a biomimetic chondrogenic microenvironment and structure of the cartilage layer for supporting cartilage regeneration, (ii) a biomimetic osteogenic microenvironment and structure of the bone layer for supporting bone regeneration, and (iii) a biomimetic interface between the cartilage and bone layer similar to the native osteochondral interface. 11his review summarizes osteochondral defects, materials that can be used for healing these defects, and in vitro and in vivo evaluation of these materials.
involves the dissolution of crystalline hydroxyapatite and proteolytic cleavage of the calcified extracellular matrix composed of organic molecules and hydroxyapatite, which is rich in collagen. 27one resorption is necessary for many skeletal processes.It is an obligatory event during bone growth, tooth eruption, and fracture healing, and it is also necessary for the maintenance of an appropriate level of blood calcium.It is a complex process involving highly coordinated interactions between osteoblasts and osteoclasts that are modulated by a system composed of the receptor activator of nuclear factor-kappa B (RANK), the RANK ligand (RANKL), and osteoprotegerin (OPG). 27steoclasts, the cells responsible for bone resorption, can be regarded as a prototype of osteoimmune cells.Typical osteoimmune disorders (e.g., rheumatoid arthritis, osteoporosis) that are characterized by bone erosions in multiple joints in conjunction with inflammation of the synovium, are stimulated by osteoclasts. 28,111he resorption of bone consists of a multistep procedure requiring attachment to the bone surface, remodelling of cytoskeletal structure, polarization of the membrane, and the transport of vesicles. 29.3.Osteochondral Defects.The development of an osteochondral defect is caused by trauma, disease, or aging. 30It is driven by a serious factor mainly in response to an inflammatory environment, e.g., inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor α (TNF-α).The local inflammation has been proved to act as a barrier to the osteogenic and chondrogenic differentiation of MSCs. 23At the same time there is an increasing production of reactive oxygen species (ROS), which subsequently contribute the secretion of matrix metalloproteinases (MMP) and ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs). 31,112Both oxidative stress and inflammation can be involved in the development of osteoporosis by preventing the differentiation of osteoblasts, inducing the differentiation and activity of osteoclasts, enhancing apoptotic osteocytes, and increasing the expression of RANKL and the RANKL/OPGratio. 113However, during the inflammation collagen II is degraded, which is the main component of hyaline cartilage, and this impairs cartilage function and increases osteoclastic activation, which reduces bone healing. 114,115he most important studied growth factors involved in osteochondral defects are transforming growth factor (TGFβ), bone morphology factor (BMP), insulin growth factor (IGF), and fibroblast growth factor (FGF), 9 which are involved in bone and cartilage formation and regeneration.The role of vasculature in bone and osteochondral development, growth and repair are well documented. 32asculature has been found to promote the expression of osteogenic genes in hypertrophic chondrocytes, thus promoting the initiation of the chondrocyte to osteoblast transformation.
Normal cartilage contains no blood vessels.In osteoarthritis, cartilage angiogenesis is involved in osteophyte development, subchondral bone remodelling, and cartilage mineralization.Osteoblasts and osteoclasts express vascular endothelial growth factor (VEGF) and its receptors, which are important during angiogenesis and bone remodelling.Blood vessel development at the osteochondral junction could increase osteochondral ossification.Pro-angiogenic factors VEGF and FGF were detected in normal cartilage (despite the fact that there are no blood vessels present). 33Inflammation can trigger angiogenesis.
During osteoarthritis, chondrocytes overproduce matrix metalloproteinases, and there is an increased production of collagen I and III and nonspecific alkaline phosphatase.Osteoblasts are characterized by an increased production of collagen I, interleukin 6 and 8. 33 This part combines the main characteristic of cartilage and bone and forms an interface between them.

MATERIALS SUITABLE FOR HEALING OSTEOCHONDRAL DEFECTS
Scaffolds are a crucial component of tissue engineering, because they offer a three-dimensional structure for cell adhesion and growth. 116Scaffolds are designed using a wide range of synthetic or natural polymers.In contrast to artificial materials and structures, biomaterials have biological traits that replicate the original type of tissue, enable favorable signal transduction, cytocompatibility, and biodegradation.Some of the materials used for regeneration are composed of a general polymer enriched with some bioactive compounds. 34Bioactivity was defined by Larry Hench as a material property that leads to the formation of a very strong bond between biomaterials and bone tissue. 35Hierarchical materials are beneficial for material−cell interaction, as they mimic the hierarchical structure of tissue.Biomaterials for the construction of multiphasic osteochondral scaffolds are generally used to make a mineral-containing layer for bone regeneration and a polymer layer for cartilage regeneration. 7,117The most frequently explored materials for osteochondral tissue are synthetic: e.g., polyethylenglycol (PEG), polyvinyalcohol (PVA), polyamino acid (PAA) 65 or natural polymers, e.g., chitosan, hyaluronic acid, chondroitin sulfate, alginate, silk, and gelatin. 7,393.1.Synthetic Polymers.Polycaprolactone (PCL) has been widely used in tissue engineering for the fabrication of bone scaffolds due to its excellent biocompatibility, slow degradation (2−3 years), and mechanical properties. 36,118owever, it has low bioactivity and needs to be supplied with some bioactive additives, e.g., hydroxyapatite (HA), tricalcium phosphate, and bioactive glass (BG), 37 or ceramic additives. 38olypept(o)ides or poly(amino acid)s (PAA) are formed by the ring-opening polymerization of amino acid N-carboxyanhydrides (NCA) and have the benefits of natural and synthetic polymers.Yang et al. prepared the material PAA-RGD (thiol/thioester dual-functionalized hyperbranched polypeptide P(EG3Glu-co-Cys) and maleimide-functionalized polysarcosin, which promote the proliferation and chondro-genesis of MSCs and produce ostechondral repair in New Zealand Rabbits. 39oly(lactide-co-glycolide) (PLGA) is a very useful biodegradable polymer due to its tunable biodegradation rates and very good mechanical and elastic properties, 40 with a faster degradation period than PCL. 36It has been used for preparing a bilayered scaffold with a different pore size and porosities in the chondral (100−200 μm) and osseous layer (300−450 μm).The combination of PLGA and β-TCP exhibited very good biocompatibility, osteinductivity and biodegrability both in vitro and in vivo. 41oly(vinyl alcohol) (PVA) is a synthetic polymer used in tissue engineering scaffolds for its ease of fabrication. 42revious reports demonstrated that PVA is suitable for the fabrication of 3D-printed scaffolds with sufficient physical stability for bone tissue engineering.PVA has a unique adhesive function which can interact with components in the ECM.PVA exhibited hydrophilicity, permeability, biodegradability, and biocompatibility.It is able to retain a large amount of water or biological fluid without dissolving. 43elatin-methacrylate (GelMA), a derivate of gelatin, possesses a large number of arginine-glycine-aspartic acid sequences that are favorable for cell adhesion, migration and growth. 11,44,119It has excellent biocompatibility and enzymatic degradation. 45Pure GelMA has poor mechanical properties and a relatively rapid degradation rate. 46Moreover, GelMA exhibits superior printability properties compared to gelatin and other bioinks. 47GelMA hydrogel, prepared by optical cross-linking, has advantages such as injectability and low toxicity. 62Yang et al. prepared a PAA-based hydrogel (PAA-RGD) that promoted the proliferation and chondrogenesis of MSCs. 39Gellan gum is a calcium-cross-linkable polysaccharide hydrogel that could be used for cartilage/bone regeneration due to its structure being similar to the natural glycosaminoglycan present in cartilage, and its high affinity for calcium. 66urthermore, conductive hydrogels comprise a water-soluble polymer and a network infused with conductive materials, e.g., carbon nanotubes, graphene oxide, or a conductive polymer.These materials impart electrical stimuli to the wound site and support the migration, proliferation and differentiation of cells and angiogenesis. 120Graphene oxide (GO) is produced by the oxidation and exfoliation of graphite.It contains abundant oxygen-containing groups such as carboxyl, epoxy, carbonyl, hydroxyl, etc. 58 GO exhibits an efficiency for anchoring calcium ions and increasing mineralization, differentiation, and cell proliferation.GO can act as an enhancer of the mechanical, electrical and cellular properties of a hydrogel. 62It is able to interact with different polymers and support bone wound healing 63 and promote bone tissue regeneration 64 through biomineralization that accelerates the formation of calcium phosphate crystals, including HAP. 58 3.2.Natural Polymers.Natural polymers are generally better than synthetic polymers in terms of cytocompatibility and bioactivity, but relatively weak in terms of mechanical properties and degradation. 121−51 Chitin, collagen, and chitosan are the most widely employed natural polymers for medical applications, especially in bone tissue engineering. 9,48,51A previous study demonstrated that photo-crosslinked alginate hydrogels were able to repair bone defects by delivering osteogenic materials. 49A silk-based hydrogel with CuTa nanozyme (i.e., CuTa@SF hydrogel) combined tannic acid (TA) and copper nanoparticles. 31It has been reported that Ta, with its antioxidant and anti-inflammatory properties, is able to decrease the intracellular ROS level, and the copper accelerated cell proliferation, thereby promoting tissue regeneration.The materials suitable for osteochondral regeneration are summarized in Table 1.

Other Components of Scaffolds. 3.3.1. Tricalcium Phosphate. β-Tricalcium phosphate (β-TCP
) is a synthetic ceramic with a chemical composition close to the mineral phase of bone. 52It was reported that TCP has osteoinductive properties and bioresorbality, and provides a reasonable template for the formation of new bone. 53Several studies have investigated the potential of bioactive glass for improving the properties of β-TCP ceramics, including high porosity and interconnectivity, favorable pore shape, appropriate pore size, sufficient mechanical strength and superior bioactivity. 54A difference in β-TCP porosity was reported to influence its mechanical durability and the length of time until artificial bone blocks are replaced with native bone tissue. 55.3.2.Bioactive Glass.Bioactive glass (BG) is a ceramic material that can be incorporated into the polymer matrix to create a nanocomposite with high potential for biomedical application.It is able to form a bond between soft and hard tissue and enhance stiffness.119 Its great osteoinductivity and osteoconductivity to stimulate osteoblast activity has been demonstrated.43 Mesoporous bioactive glass is extensively studied in bone healing due to its good biocompatibility and bioactivity�in vitro mineralization.8 Controlled ion release from bioglass is critical for osteogenesis.Also, the cellular activity of ALP (an early marker of osteoblast differentiation) increases with the amount of bioglass.35 The pore size of bioglass loaded with BMP-2 is in the range of 300−500 μm, which is important for mineralization, due to the large amount of calcium-phosphate deposition.56 Elements such as calcium, phosphorus, and magnesium liberated from the bioceramic composition create a microenvironment that is similar to the native microenvironment in situ, thus stimulating bone reconstruction and neoangiogenesis.54 3.3.3. Hydoxyapatite. Th mineral hydroxyapatite (HAP) is naturally synthesized and comprises 70% of the skeleton by weight and 50% by volume, 4 and 90% by weight of tooth enamel.HAP consists mainly of calcium and phosphate Ca 5 (PO 4 ) 3 (OH) (or more often referred to as the two units' crystal form: Ca 10 (PO 4 ) 6 (OH) 2 ) in a ratio of 1.67), and is crystalline in form.0 Nanohydroxyapatite (n-HAP) breaks down in a decomposition process to produce water and organic and inorganic compounds essential for bone healing.4 Nanosized hydroxyapatite, which is generated endogenously by osteoblasts in the form of matrix vesicles as the initiator of bone formation in the skeleton, has great osteoinductive properties.131 As well as playing a role in the pathological calcification of cartilage and vasculature, it can be deposited in soft tissues in the form of dystrophic and metastatic calcifications.19 n-HAP is a bioactive compound that can form a strong bond with bone, it is able to deposit in bone and react with proteins, resulting in an osteogenic process.58 However, the available results indicate that pure HAP has poor mechanical properties and cannot be used as a load-bearing implant material, which is forcing materials scientists to search for HAP composites with high load-bearing capacity.59 The combination of ossein and hydroxyapatite forms a complex with osteocalcin and type collagen I 60 (Osteogenon, Osteo, Pierre Fabre).This bioactive material supports cell proliferation, adhesion and bone mineralization.61 It has two effects on metabolism: it inhibits osteoclasts and stimulates osteoblasts.It has been applied in tablet form for its analgesic effect and for reducing bone loss and the chance of fracture in patients with secondary osteoporosis.It is more effective at preventing bone loss than other calcium salts.

Fabrication Technique.
Fabrication technique plays a crucial role in the development of new materials, and consequently has an influence on repair outcomes by modulating biological responses through controlling pore structure, mechanical properties, the spatial distribution of materials and/or additives such as cells and growth factors, and biodegradation.−135 Extrusion printing is the most commonly used method for constructing 3D architecture in osteochondral tissue engineering studies because of its relatively low cost, high availability, and ease of use. 1413D printing technology, also known as additive manufacturing, allows precise control over the composition and spatial distribution of cells and biomaterials to create scaffolds for tissue repair and regeneration. 51It involves stacking layers of biologically active materials filled with cells and growth factors to create highly biomimetic tissue microenvironments, structures, blood vessels, and functional artificial organs. 138Evolving 3D bioprinting approaches have resulted in a number of different printing strategies, including free-form reversible embedding printing of suspended hydrogels, extrusion-based multimaterial point-dispensing printing, void-free 3D bioprinting, and layer-by-layer alternating bioprinting with a double mesh for tissue analogues.As tissues contain multiple structures and diverse cells, and cell phenotype is sensitive to the biochemical and mechanical properties of the microenvironment, bioinks for 3D bioprinting are becoming increasingly important for the generation of multiple structures and diverse cell-engineered tissues.This enables the production of patient-specific tissues with the correct size and shape. 13,139,140

EVALUATION OF MATERIALS FOR THE TREATMENT OF OSTEOCHONDRAL DEFECTS: IN VITRO AND IN VIVO METHODS
This review aims to present the most widely used methods for evaluating materials for the treatment of osteochondral defects.Special attention will be paid to in vitro (e.g., cytotoxicity, biocompatibility) and in vivo methods (e.g., histological staining, CT etc.), which enable systematic and comprehensive testing of these biomaterials in the laboratory and clinical environment (Figure 2).4.1.3.AK Test.(Adenylate kinase): the release of adenylate kinase from cells can be an indicator of cell damage and cytotoxicity caused by a biomaterial. 69.1.4.Live/Dead Staining.Fluorescent dyes such as acridine orange, fluorescein diacetate, and propidium iodide are used to visualize live and dead cells on the surface of a biomaterial under a microscope.8,70 4.1.4.1.Alexa Phalloidin Cytoskeleton and DAPI Nuclear Staining.Alexa phalloidin cytoskeleton and DAPI nuclear staining are used to determine cell adhesion, proliferation and biocompatibility on biomaterials.8,71 4.1.5. Alkane Phosphatase (ALP).ALP is an enzyme associated with the differentiation of osteogenic cells, and plays a key role in bone mineralization processes and has a critical function in the formation of hard tissue.72 Higher ALP activities were indicated in cells cultured on the biomaterial, demonstrating a positive effect of the biomaterial on osteogenesis.73 During the osteogenic differentiation process, the presence of ALP activity indicates the differentiation of mesenchymal stromal cells (MSCs) into osteoblasts.74 This can be determined spectrophotometrically, by RT-PCR, Western blot, or immunostaining, 75−77 4.1.6.Bone Morphogenetic Proteins (BMPs) and TGF-β Family.BMPs are multifunctional growth factors that belong to the transforming growth factor beta (TGF-β) superfamily.78 TGF-β is a multifunctional chondroblast growth factor that promotes the secretion of collagen II and proteoglycans.It also plays a critical role in maintaining of homeostasis between subchondral bone and articular cartilage.138 BMPs are involved in several events during bone morphogenesis, including bone remodelling, bone formation, chondrogenesis, and mesenchymal cell infiltration and proliferation.79,80 It is mostly determined by ELISA, RT-PCR, or immunocytochemistry. 81 4.1.7. Ostcalcin (OC).OC is a calcium-binding protein, and is the most abundant noncollagenous protein in bone.82,83 OC is a marker of osteoblast mineralization, and can reflect their activity.Its determination is important for assessing the ability of biomaterials to support osteogenesis.Like other osteogenic factors, it can be determined using RT-PCR and immunocytochemical staining or ELISA.84−86 4.1.8. Rut-Related Transcription Factor 2 (RUNX2).Runx2 is a master gene of osteoblast differentiation and bone formation.87 It is most often determined using RT-PCR 88 or immunocytochemistry. 89 4.1.9.Collagen I/Collagen II. Nuerous collagen subtypes have been identified in articular cartilage, such as types I and II.Collagen type I is the main component in connective tissues 90 and is also the main organic component of the bone matrix produced by osteoblasts.The presence of type I collagen is necessary for the formation and proper regeneration of bone tissue around an osteochondral defect.In contrast, collagen type II is produced in cartilage by chondrocytes, and is a key factor in restoring the structure and function of cartilage tissue.91 They are most often determined using the ELISA method, 92 RT-PCR 93 or immunocytochemistry. 94 4.1.10. Agecan and SOX9.Aggrecan and SOX9 are the main markers in cartilage that characterize chondrogenic differentiation during cartilage formation.Aggrecan contains several highly sulfated GAG side chains that confer a high negative fixed charge density to the tissue, providing hydration and compressive stiffness. The amount of aggrecan/SOX9 produced by chondrocytes can be detected by RT-PCR or immunocytochemistry. 93,[95][96][97][98][99][100]121,140 Once the in vitro (Figure 2A) tests are completed, it is important to perform an in vivo (Figure 2B) evaluation of the biomaterial for the treatment of osteochondral defects. He are some possible methods for the in vivo assessment of materials.
4.2.In Vivo Methods.4.2.1.Histological Staining.Histological staining is a method that highlights important features of the tissue. 101The tissue sections are stained with hematoxylin and eosin for nuclear morphology and nonspecific tissue visualization, alcian blue to assess sulfated glycosaminoglycans content, picrosirius red to assess collagen content, and alizarin red to assess calcification. 102−105 4.2.2.Microcomputed Tomography (micro-CT) Analysis.−108 3D evaluation is carried out on the segmented images to determine bone volume and density and to reconstruct a 3D image.
Micro-CT images are further analyzed using ImageJ to determine trabecular thickness (which quantifies bone growth), bone volume to total volume ratio, porosity, and the degree of anisotropy within the tested samples, offering insight into the regeneration of the subchondral bone. 109

THERAPEUTIC APPLICATION AND COMMERCIALIZATION OF THE PRODUCT
Currently, the repair of osteochondral defects is one of the most difficult challenges in medicine.Before a potential treatment is applied to humans, all the necessary preclinical experiments need to be conducted (both in vitro and in vivo).
In the subsequent clinical evaluation, there is a set of ongoing activities that use scientifically sound methods for the assessment and analysis of clinical data to validate safety, clinical performance, and effectiveness. 136For clinical applications, it is essential to include convenience, effectiveness and minimize trauma during the application process.Thus, the choice of biomaterials is extremely important and needs to be not only considered for its chemical composition, but also its physical properties. 137

CONCLUSIONS AND FUTURE OUTCOME
This review summarizes materials suitable for osteochondral regeneration, and the second part of this review is concerned with in vitro and in vivo methods necessary for the evaluation of new materials and therapeutic applications.Ideally, autologous tissues (autografts) are used for medical applications, which eliminates the immunogenic response and is optimal for cell growth.Alternatively, grafts from donors (allographs) can be used, but there is the risk of immunoreaction and infection.Furthermore, these strategies are based on cellular techniques, and these products were considered as medical devices and biological medicines that need time-consuming and expensive authorization.Thus, nowadays, the majority of materials for osteochondral regeneration usually consist of synthetic or natural polymers.Some of these polymers have low biological activity, and need to be supplemented with substances with higher biological potential, e.g., β-TCP, GO, etc. Due to a wide range of biocompatibilities and biodegradabilities, these could have a different influence on the surrounding tissue and potential inflammation.
Osteochondral tissue is a complex structure with multiple hierarchies.The new material has to incorporate characteristic properties of each part of osteochondral units, e.g., porosity, the production of ECM proteins (collagen I for the bone part and collagen II for the cartilage part).The regeneration of a seamless gradient between the innervated, vascularized, and mineralized bone, and the avascular, nonmineralized, and aneural cartilage should be one of the most important tasks in osteochondral tissue regeneration.Given the diverse material composition of the indigenous osteochondral tissue, it is essential to select suitable biomaterials for each layer.Due to the complicated structure of the osteochondral part, it is difficult to find materials that perfectly substitute for a defect.This is a reason why multilayered systems are so popular.However, gradient scaffolds due to their different porosities mimic the hierarchy of the natural tissue, which plays an essential role in nutrient and oxygen transport, cell adhesion, and migration and vascular growth.
The following are recommended for the preparation of new materials: Improving the biocompatibility of scaffolds.Aiming for good mechanical properties and an ideal biodegradation rate.
Focusing on promising 3D-printed methods that can match the shape of the damaged part.
Optimizing transplantation (preclinical studies) to avoid the rejection of materials.
So, the major challenges are to find acellular materials that reflect the osseous and chondrogenic part, mimic the gradient microstructure, have appropriate mechanical properties, an appropriate degradation profile, and include all the requirements for healing an osteochondral defect.

Figure 1 .
Figure 1.An osteochondral defect that includes both cartilage and subchondral bone damage.During healing, the main cells of cartilage− chondrocytes and bone−osteoblasts, release/express characteristic biomarkers."Created with BioRender.com".

4 . 1 .
In Vitro Methods.First of all, it is important to determine cytotoxicity and biocompatibility in vitro.There are several established methods for this: 4.1.1.MTT Test.(3-(4,5-Dimethylthiazol-2 yl) 2,5-diphenyltetrazolium bromide): this staining method measures cell viability in the presence of a biomaterial.Cellular mitochondria convert MTT to a purple formazan precipitate, which can be quantified spectrophotometrically. 8,67 4.1.2.LDH Test.(Lactate dehydrogenase): this measures the release of the LDH enzyme from cells, which is an indicator of cell damage.Elevated LDH levels indicate the cytotoxicity of a biomaterial.68

Figure 2 .
Figure 2. (A) In vitro assessment of biomaterials (determination of cytotoxicity, biocompatibility and selected biomarkers) and (B) evaluation of biomaterials by in vivo methods, using histological staining and micro-CT."Created with BioRender.com".

Table 1 .
Summary of Materials Used for Healing Osteochondral Defects Included in This Review a