The current fast lifespan emphasizes the need to bring a new trends and challenges in the treatment of bone and cartilage defects, as well as the development of new healing methods and biomaterial substitutes of cartilage and bone in view of minimally invasive surgical procedures and enhancing of healing process in affected site. From the point of view biomaterials, the first requirement represents the optimal filling of irregular and complex-shaped defects in the human population (Mano and Reis, 2007). Damage in the form of individual chondral or osteochondral lesions accounts for 67% of all arthroscopies (Widuchovski et al., 2007) and the injury of the articular cartilage, inflammatory and non-inflammatory diseases, irregular cartilage surface, symptomatic full-thickness cartilage lesions or degeneration of the cartilage results in the destruction of the normal chondral architecture as well as the bone structure in affected area and finally causes the osteoarthritis (Maglio et al., 2019). The osteoarthritis is a degenerative joint disease with typical symptoms (redness, swelling, heat, effluent, painfulness), loss of articular cartilage, narrowing of the joint space, inflammation, changes in synovium and bone structure (subchondral bone remodeling, cysts or osteophytes formation, sclerosis and lesions of the bone marrow (Macfadyen et al., 2019). However, early diagnosis by conventional radiography (X-ray), which allows indirect evaluation of damaged cartilage and bone, or imaging of hyaline cartilage by non-invasive method, direct magnetic resonance imaging (MRI examination), are good choices for capturing joint damage, traumatic or degenerative cartilaginous lesions in terms of current therapeutic options (Rodrigues and Camanho, 2010). In the body, the knee joints belong to the most frequently affected joints. The permanent damage of the articular cartilage and subchondral bone (osteochondral defects) represents an interesting opportunity for the scientists and clinicians to explore new methods or special materials for regeneration of osteochondral defects (Chu et al., 2010). The surgical approaches as mosaicplasty, arthroscopic microsurgery, chondrocyte implantation, or others, which were used for chondral or osteochondral defects therapy frequently failed due to incomplete fibrous or fibrocartilage tissue formation (Ozmeric et al., 2014). Currently, there are many methods and processes using different materials for treatment of cartilage lesions or bone. Meng et al. (2020) described in their review article different animal models, as a small, laboratory animals (rat, rabbit) and large, domesticated animals (dog, pig, sheep, goat, horse) of the osteochondral defect and possibility of the treatment using of the different biomaterials ((osteochondral allograft, different types of scaffolds with MSC, transplantation/autologous cartilage chips, bone marrow derived cell concentrates, Hyaluronic acid in various forms). Authors used several locations (trochlear and patellar groove of the femur, femoral condyles) and different endpoints of the treatment (from 6 weeks to 24 months). The research of the osteochondral defects in rabbits with using of combination of biomaterials (chitosan) and cartilage promotive drug (icariin-conditioned serum) showed regenerative potential of this treatment method (ZhangMing et al., 2020). The suitable scaffolds for clinical use should be biodegradable, mechanically stable, noncytotoxic and ought to temporarily function as a support for the cells or other additives to facilitate the appropriate repair processes (Dhollander et al., 2012). Cement materials on the base of calcium phosphate have many interesting properties such as biocompatibility, bioresorbability, moldability, ability to support osteoconduction, osteoinduction and osseointegration, absence of any by-products during the process of metabolism and big potential for the replacement by new chondral and bone tissue after orthopedic surgery (Ambard and Mueninghoff, 2006). Scaffolds on the base of calcium phosphate biocement was studied in a bone structure regeneration, where it exhibited excellent biocompatibility and osteoconductivity (Cancedda et al., 2007), a similar type of biomaterial on the same base was described in small laboratory animals like a rabbit for treatment of chondral lesions on femoral condyle (Bernstein et al., 2013). The healthy articular cartilage is characterized by a smooth, glistening, bluish durable surface, and is largely responsible for the absorption of impact forces, and the ability to withstand high compressive forces. It has the ability to manage weight bearing with support of subchondral bone and facilitates bone movement (Junqueira and Carneiro, 2005; Maglio et al., 2019). The subchondral bone composed of water, hydroxyapatite and glycoproteins (type I and V collagen) is responsible for bone outer shape and appropriate biomechanical conditions for the development of cartilage at the physiological conditions in the knee joint. The articular cartilage is well integrated with underlying subchondral bone. Therefore, during the therapy of the chondral defects this bone structure should not be neglected (Deng et al., 2019). The hyaline cartilage is highly hydrated and specific connective tissue due to its composition. It does not have its own blood supply, network of nerves or lymphatic drainage (Reinholz et al., 2004; Malda et al., 2019). Experimental, preclinical animal models with artificially created osteochondral lesions that were studied include laboratory animals (Bauer et al., 2017), dogs (Salkeld et al., 2016), small ruminants (Yucekul et al., 2017) and horses (Vindas Bolanos et al., 2017). The healing of the osteochondral defects in animal models is significantly influenced by differences in metabolism, diet, type of the digestive system and the levels of specific hormones relative to the levels in humans (Jainudeen et al., 2000). The selection of a suitable animal model is a prerequisite for connection between development of the new biomaterials and their application in clinical practice as well as for the proper evaluation of a degradability, biocompatibility, and interaction of biomaterials with cartilage and subchondral bone (Meng et al., 2020). It seems that the pig model (Large white pig) represents a more appropriate animal model for the study of osteochondral defect, compared to humans, because the pigs have similar anatomical structure of the organs. The size of the porcine joints, their weight, and thickness of the articular cartilage are very similar to the human population compared to another animal model (Meng et al., 2020); consequently, the pig model was used in this work. The porcine cartilage thickness is 1.5–2.0 mm thick Ahern et al., 2009) in the medial femoral condyle, while in humans, the thickness varies from 1.65 to 2.65 mm (Shepherd and Seedhom, 1999). The smallest chondral lesion that requires treatment in humans should be at least 10 mm in diameter (Chu et al., 2010). The cartilage defect with critical size of 6 mm heals spontaneously in the large pigs (Ahern et al., 2009). The purpose of this study was to investigate the potential of acellular calcium phosphate biocement in the regenerating process of the created osteochondral defect (diameter of 10 mm, depth of 10 mm) on the medial femoral condyle in large pigs. The repair ability of calcium phosphate biocement was compared to spontaneous healing of an artificial created cyst and natural cartilage and bone structure.

The complex physico-chemical properties of CAL cement were discussed in the previous article (Medvecký et al., 2021). Note that the analysis of properties clearly showed a significant reduction in compressive strength of the cement (down to 5 MPa in the wet state) after the addition of amino acids compared to tetracalcium phosphate/monetite biocement without additives. In addition, a large amount of amino acids remained adsorbed on the surface of the formed nanohydroxyapatite particles in the cement, which effectively modified the morphology of its particles from needle-like to spherical, which is more suitable for cell adhesion and growth. The presence of amino acids in the cement suppressed the increase in pH during setting of the cement, demonstrating the suitable buffering capacity of the amino acids in the cement. In addition, the porosity of CAL cement was relatively high (about 57%) and two pore fractions of different sizes were identified in the cement microstructure - larger micropores with a size of 10–15 µm and a main fraction with 1–3 µm micropores. Similarly, RT-qPCR analysis showed high stimulation of osteoblasts to express of the collagen I, OP, ON and ALP in CAL.In terms of collagen II production, the addition of glycine with a small supplementation of LYS or PRO in culture medium strongly induced the in vitro production of collagen II by chondrocytes (De Paz-Lugo et al., 2018). On the other hand, the PRO supplementation of medium had little but significant effect on in vitro collagen biosynthesis by fibroblasts, but nevertheless a strong positive effect of extracellular PRO on collagen biosynthesis was verified in case of insufficient endogenous PRO synthesis (Karna et al., 2020). We hypothesize that the mixture of amino acids in calcium phosphate cement promotes the differentiation of MSCs into chondrocytes by increasing collagen biosynthesis due to reduction in further endogenous synthesis of the corresponding amino acids required for collagen production by cells. In addition, the reduced mechanical properties of cements contribute to the differentiation of bone marrow stem cells into chondrocytes and the subsequent formation and growth of cartilage tissue.

The knee joint is the supporting structure of the pelvic limb. It resists tremendous impacts and loading of the articular cartilage and subchondral bone, but after long time, these factors can result in cartilage and bone tissue damage and may lead to the development arthritis or osteoarthritis. Many previous studies have reported that newly formed cartilage rarely has the same biomechanical properties as physiological hyaline cartilage. In the case of the deep osteochondral defects where the subchondral bone has been damaged, treatment strategies of those structures should also be considered (Interna et al., 2011). There are previous studies, which noted that the newly formed bone has the similar biomechanical properties as surrounding bone tissue, because the bone is dynamic structure getting constant remodelling. Each restoration of the fracture or bone defects treatment depends on using material treated area of the body and very important role has a type of ossification (Kim et al., 2020). Autogenous bone grafts constitute as a golden standard in therapy of the bone tissue (Wang and Yeung, 2017). Excellent properties like as osteoconductivity and osteoinductivity of the implants based on calcium phosphate as evidenced our results. In the osteochondral defect, the biocement paste CAL promoted a bone formation, remodelling and osteointegration, leading to enhanced repair of the subchondral bone defect. Therapy of deep osteochondral defects of the knee joint presents a big challenge for in vivo research aimed at optimizing healing techniques and methods (Lydon et al., 2019; Kresáková et al., 2021). In the current time between the normally using healing therapies of the stifle joint articular cartilage after the traumatic damages belong microfractures and the osteochondral autografts and allografts (Mathis et al., 2018). The treatment should include only minimally invasive surgical procedures and methods of marrow cells stimulation to support the progress of the formation of new tissue with the same properties as the physiological chondral tissue and bone structure (Carneiro et al., 2013). Scientists have been researching the role of different materials in the treatment of osteochondral lesions for many years. The properties of the materials can greatly influence of the mechanical environment of the healing process in treatment osteochondral defects (Davis et al., 2021). It is possible to study various implants on the experimental animal models under physiological conditions, as they provide better understanding of the response in humans. Our study compared results from a treatment of the artificially created osteochondral defects (10 mm diameter and 10 mm depth) with special biocement and spontaneous healing of the artificial cysts with control healthy cartilage and subchondral bone, in the medial condyle of the left knee joint in the pig within 3 months. Ho et al. studied porcine osteochondral defects in two areas, in the high-load bearing femoral condyle and in the low-load bearing trochlear groove. They found that better results were in the condyles, because mechanical stimulus had beneficial effects on the regeneration of the chondral and bone tissue. This study pointed to a close interaction between the articular cartilage and bone tissue, because the treatment of chondral defect was dependent on the underlying subchondral bone (Ho et al., 2010). The other investigation affirmed that differences of biomechanical properties between artificial tissue, biomaterials and subchondral bone in different animal species can be the reason for incomplete healing created defects (Archibald et al., 2002). Therefore we choose the medial condyle of femur for creation of the osteochondral defect in our study. Prior research has demonstrated that the use of the porcine model is more comparable with publications, which described other species of animals as a model (Ahern et al., 2009; Pilichi et al., 2014; Vilela et al., 2015; Kresáková et al., 2021). Pearce et al. (2007) stated that the bone composition, micro- and macrostructure, remodelling and the bone density in the pig is very similar to that with humans. Shimomura et al. (2010) and Archibald et al. (2002) affirm that due to the thickness of the porcine articular cartilage on medial femoral condyle being comparable with chondral layer in humans, this animal model should be used for the study and evaluation of the healing the chondral or osteochondral defects. The thickness of the articular cartilage changes depending on the age and varies among the species. The younger animals have thicker cartilage layers than the older ones, but the content of GAG and flexibility are lower in immature cartilage. The biomechanical properties of the chondral structures are influenced by the content of GAG too (Shimomura et al., 2010). As evidenced by our results of staining, the highest intensity of Safranin Q (the proof of the highest content of proteoglycan) was found in the radial zone. The properties of the chondral cells (morphology, density) and another component in all layers were comparable with hyaline cartilage. Our study confirmed that the subchondral bleeding strategy, with drilling marrow stimulating techniques, is very important for the formation and repair of the chondral and bone tissue, because the blood from the bone marrow contains mesenchymal stem cells, which differentiate into osteoblasts and chondroblasts. On the similar principle functions the surgical method of the microfractures, where is recruitment of stem cells by drilling the subchondral bone to allow bone marrow to flow into the osteochodral defect site and initiate healing (Yen et al., 2008). Many previous studies pointed out, that the bone marrow stimulation induced the bleeding, which in the defect site supported a blood clot formation, composed of the mesenchymal stem cells (MSC) that facilitated tissue regeneration and served as template for the matrix formation (Smith et al., 2005; Madry et al., 2017; Zlotnick et al., 2021). This is also evidenced by other research results that consisted of cell tracing using iron oxide particles in cartilage defects without drilling into the subchondral bone. This research found that approximately 30% of the cells in the new cartilage tissue at 12 weeks were coming from the transplanted population, because the majority of cells migrated from subchondral bone (Blanke et al., 2009). Many different chondral lesions have been treated by the implantation techniques using chondrocytes, but these treatments failed, because mechanical stability was not provided (Matricali et al., 2010). Other authors noted, that after using poly-LD-lactic scaffold or polycaprolactone nanofibrous scaffold with seeded cells (chondrocytes, MSCs), were repaired only defects of cartilage, but bone structure did not have properties of the subchondral bone. There was only soft tissue or cartilage instead of bone structure (Pulliainen et al., 2007). In scaffolds seeded by cells, a major problem is hypertrophy of these cells (Bernstein et al., 2013). Currently, the osteochondral damage therapy focuses on replacement of damaged cartilage and bone by special biomaterials, which lead to regeneration of the chondral and bone tissue (Kon et al., 2012). According to some authors, the part of scaffolds that remained in bone neither degraded nor slowed down the treatment process (Leja and Lewandowicz, 2010). The macroscopic, microscopic assessment and radiographic analysis demonstrated the successful regeneration of the treated osteochondral defects in a porcine knee joint using cell free calcium phosphate scaffold CAL in our research. Both types of tissues in the site healed osteochondral defects had typical features of the chondral and bone tissue. In comparison, several publications described artificial scaffold, which can be inappropriate to regenerate articular cartilage, or only lead to fibrocartilage formation (Higa et al., 2017; Murata et al., 2018; Tamaddon et al., 2020). The results of some experiments indicate that combination of special additives (erythropoietin, bone marrow aspiration concentrate) with scaffold could help improve the healing of osteochondral defects (Betsh et al., 2014). The basis of many studies of tissue engineering methods was bone marrow cells collection, isolation, cultivation and their application separated or embedded in biomaterials into the created defects. Each of these steps required at least two surgery procedures (Liua et al., 2017; Bozkurt et al., 2019). According to the results of the cartilage biomechanical research of Archibald, et al., pigs, as animal models used for creation of chondral or osteochondral defects, have 2–5 times stronger and stiffer subchondral bone than humans do (Archibald et al., 2002). Our objective was to only employ a single surgical procedure, where we created a full-thickness osteochondral defect, based on deeper drilling to the subchondral bone, allowing for the supply of the blood from the bone marrow, together with its individual components necessary for regeneration processes. We also analyzed the results of our study with radiographic methods, which confirmed the findings of the previous evaluations (macroscopic, histological) of treated knee joints. In the animal group treated with biomaterial CAL, the defect filled by the new regenerated chondral and bone tissue and biocement was almost fully reabsorbed.

In conclusion we can state, that the important fact is that chondral and bone tissue are closely related. Therefore, during the treatment of the osteochondral defects, they must be considered as a complex of articular cartilage and subchondral bone, which have a very well organized tissue. The further research of the osteochondral lesions therapy should focus on improving modification of scaffolds, which will have the ability to create the physiological architecture of regenerated cartilage and subchondral bone, supporting their functional character and their mechanical properties. Although the bone marrow cells, growth factors and cytokines in scaffolds are important and interesting for the right repair processes in osteochondral lesions, their difficult storage, low survival rate of cells and instability of factors, makes use of the cell-free scaffolds for the therapy in the cartilage and subchondral bone preferable.