Abstract
Mesenchymal stem cells (MSCs) from bone marrow (BM) are widely used for bone and less for cartilage tissue regeneration due to their self-renewal and differentiating properties into osteogenic or chondrogenic lineages. This review considers the last decade of clinical trials involving a two-step procedure, by expanding in vitro MSCs from BM, or the so called “one-step” procedure, using BM in toto or BM concentrate, for the regeneration of cartilage and osteochondral tissue defects. The following conclusions were drawn: (1) Cartilage defects that can be repaired by the two-step technique are about twice the size as those where the one-step method is used; (2) the two-step procedure is especially used for the treatment of osteoarthritic lesions, whereas the one-step procedure is used for osteochondral defects; (3) the number of transplanted cells ranges between 3.8×106 and 11.2×106 cells/mL, and the period of cell culture expansion of implanted MSCs varies widely with regard to the two-step procedure; (4) hyaluronic or collagenic scaffolds are used in all the clinical studies analyzed for both techniques; (5) the follow-up of the two-step procedure is longer than that of the one-step method, despite having a lower number of patients; and, finally, (6) the mean age of the patients (about 39 years old) is similar in both procedures. Clinical results underline the safety and good and encouraging outcomes for the use of MSCs in clinics. Although more standardized procedures are required, the length of follow-up and the number of patients observed should be augmented, and the design of trials should be implemented to achieve evidence-based results.
Introduction
D
Unlike chondral lesions, osteochondral injuries penetrate the subchondral bone and elicit an inflammatory response with hematoma, fibrin clot, and the recruitment of mesenchymal stem cells (MSCs) from bone marrow (BM). However, the composition of the cartilage repair tissue rarely replicates the structure of normal articular cartilage [4].
Therefore, acute and chronic cartilage lesions are the prime candidate for tissue engineered or cell-based approaches. Several strategies have been investigated to repair articular cartilage lesions, but the correct indications and clinical results are still being debated in the current literature [5]. Hypothetically, the ideal technique for a chondral defect repair should generate a repair tissue with biomechanical properties similar to those of normal hyaline articular cartilage. Abrasion arthroplasty, subchondral bone drilling, and microfracture techniques are marrow stimulation procedures that are aimed at perforating subchondral bone to supply blood and BM to the lesion site [6]. This results in the recruitment of MSCs, fibrin clot formation, and subsequent fibrocartilage formation. Another approach is the use of autologous or allogenic grafts and mosaicplasty (transplantation of multiple, small, autologous osteochondral grafts), which are limited by marked donor-site morbidity and the availability of such tissue. Moreover, to date, the long-term clinical data do not show consistent satisfactory results, and with the lack of applicability to large lesions, doubts remain about the quality and durability of the fibrocartilage that forms after these surgical procedures [7].
Another surgical approach that is used for treating chondral lesions is the autologous chondrocyte implantation (ACI) technique, first performed by Peterson and co-workers [1], which is regarded as the first application of a cell-engineering strategy in orthopedic surgery. ACI is performed in 2 steps: Autologous chondrocytes are harvested from cartilage, isolated, and cultured, and then, culture-expanded chondrocytes are implanted into the defect [8,9]. ACI was first used in the treatment of chondral lesions of the knee, and became increasingly popular; later, it was successfully applied to the ankle [10 –12]. The excellent durability of results, obtained by ACI over time, is well known and contrasts sharply with the long-term results reported for marrow stimulation techniques (abrasion, drilling, or microfractures), which provide a fibrocartilaginous repair tissue [13,14]. In addition, the use of tissue-engineered membranes matrix guided autologous chondrocyte implantation (MACI), composed of autologous chondrocytes grown on a scaffold, enhanced the applicability of the ACI technique, thus permitting a completely arthroscopic procedure in the knee and ankle [15]. Nevertheless, ACI and MACI have 3 main drawbacks, which may never be overcome: the need for 2 surgical operations, the limited chondrogenesis of expanded chondrocytes, and their inapplicability to articular cartilage defects in OA or rheumatoid arthritis (RA), because of the poor biological properties of chondrocytes from patients affected by both OA and RA [16 –19]. Moreover, the difficulties involved in obtaining a sufficient number of chondrocytes for autotransplantation and the donor site morbidity should not be forgotten [18]. Finally, besides the techniques mentioned earlier, extracorporeal shock waves were recently used to treat a 2×2 mm experimental cartilage defect in rats, without success [20].
In the last decade, tissue engineering and regenerative medicine techniques have improved the use of autologous adult MSCs, thanks to their simultaneous capacity to proliferate and differentiate without an immunological reaction, disease transmission, or donor-site morbidity [21]. The mechanisms of action of MSCs in tissue regeneration are related to the secretion of a number of cytokines, chemokines, and growth factors (GFs), which can improve angiogenesis, suppress inflammation, inhibit apoptosis, and stimulate endogenous repair. MSC differentiation in the desired direction may be achieved as a result of environmental, mechanical, and biological stimulation according to GFs that are present [22,23]. There are several sources of MSCs (BM, adipose tissue, umbilical cord, amniotic fluid, dental pulp, peripheral blood, and skeletal muscle, even if the recovery of MSCs from these last 2 tissues is rare), but the most common source of MSCs in the orthopedic field is BM, where a small percentage (0.01%–0.001%) of the total mononuclear cells is MSCs and their amount increases by 100–10,000 fold over several weeks in culture conditions [24 –31].
For cartilage regeneration, the MSC harvesting procedure is considered, by some authors, to be less invasive than that of chondrocytes, even if, in the standard procedure, 2 surgical steps are still required because of the need for cell harvesting and expansion before the final implantation of cells into the lesion site [17,32 –35]. Moreover, MSCs can be harvested without impairing normal articular cartilage, thus preventing donor-site morbidity. Recently, researchers have focused their attention on the use of autologous whole BM, which contains not only MSCs, but also accessory cells and GFs. This technique needs no cell selection and expansion in the laboratory, and, consequently, the transplant can be performed in one operative procedure directly in the operating room (one-step arthroscopy or surgery), without the need for a Good Manufacturing Practice (GMP) facility [19].
Since the effective clinical therapeutical potential of MSCs from BM in acute and chronic chondral and osteochondral lesions is still unclear, the aim of the current narrative review is to examine the literature concerning clinical trials of the last decade regarding two- and one-step techniques with the use of in vitro expanded MSCs, BM in toto, and BM concentrate, for the regeneration of cartilage and osteochondral defects, due to trauma or joint degenerative pathologies such as OA. Safety concerns were also evaluated.
Search Strategies
To identify the studies to be considered in the current review, a PubMed database search was performed using the following MeSH: (Bone marrow OR Bone marrow transplantation OR Bone marrow cells) OR (Mesenchymal stem cells OR mesenchymal stromal cells OR mesenchymal stem cell transplantation) AND (Cartilage OR hyaline cartilage OR articular cartilage OR cartilage disease). The searching limits were: English language, humans, and papers published from January 1, 2002 to March 30, 2012.
Results and Discussion
Over the last few years and thanks to the recognized advantages of MSCs, there has been a rapid surge in the employment of MSCs in clinical trials, ranging from the treatment of bone, to neurodegenerative diseases [36,37], skin lesions [38,39], myocardial infarction [40,41], graft-versus-host disease [42,43], immune disorders [44], cirrhosis [45], ischemic stroke [46], and hematologic malignancies [47].
Due to their ubiquity, tolerance to expansion, paracrine capabilities, and multipotency, the potential of MSCs for clinical applications in the orthopedic field is encouraging, but only a few trials describe the applications of MSC for the treatment of cartilage diseases. It has been postulated that transplanted MSCs may survive and produce cartilage probably not only because they differentiate, in situ, into chondrocytes, but also because they produce some factors inducing other resident cells to differentiate into cartilage [48].
The aim of the present review was to focus on 2 different techniques for the treatment of chondral and osteochondral defects: first, the two-step procedure that employs in vitro-expanded MSCs and then, the one-step method, which uses BM concentrate or BM in toto, in the clinical field, taking into account different aspects, such as types and dimensions of lesion, the amount of implanted cells, time for cell expansion, scaffolds used, number of patients enrolled, and follow-up, to observe whether these variables influence the clinical outcome.
From the PubMed search, 516 articles were found, but 503 of them were excluded because most of them were noninherent (379/503) regarding only in vitro and animal studies or the use of MSCs from sources other than BM or were employed for the regeneration of tissues other than cartilage, and the others (124/503) were reviews. When reading the selected 13 articles, 2 additional ones were found by screening the reference lists. Finally, a total of 15 articles are discussed in the present review regarding the use of MSCs in chondral and osteochondral regeneration (Fig. 1): 3 studies analyzed osteochondral lesions [19,49,50]; 4, OA defects [17,51 –53]; 7, partial and full-thickness articular cartilage defects [32 –35,54 –56]; and 1, OA and other cartilage defects together [57]. The term MSCs refers to MSCs isolated from BM.
Diagram of the PubMed searching procedure.
Two-step procedure
Eight authors described the validity of the two-step technique in clinical practice (Table 1) and observed that expanded autologous MSCs are an acceptable procedure for treating OA [17,52,53,57], full-thickness cartilage lesions [32,33], including kissing lesions [34] and large cartilage defects [35].
OA, osteoarthritis; pt, patient; MSC, mesenchymal stem cells; BM, bone marrow; PL, platelet lysate; CD, cluster of differentiation; MRI, magnetic resonance images; VAS, visual analog scale; IKDC, International Knee Documentation Committee; KOOS, knee injury and osteoarthritis outcome score; AC, articular cartilage; ICRS, International Cartilage Research Society.
Osteoarthritis
In 2 case reports in 2008, Centeno et al. observed, after expanded MSC implantation, an increase in meniscus and cartilage volume in men with degenerative knee OA. These 2 works were the first 2 case reports about an increase in meniscus size in 2 patients, using the same cultured protocols with platelet lysate (PL) as supplemental culture GFs and 2 different amounts of MSC (3.8×106 and 11.2×106 cells/mL). Magnetic resonance images (MRI) showed an increased meniscus and cartilage volume, the range of motion increased, and modified visual analog scale (VAS) scores decreased in the 2 case reports after a 3 month follow-up [52,53].
In an early controlled study conducted by Wakitani et al., the regeneration of articular cartilage defects in OA knees was promoted by the transplantation of autologous culture-expanded MSCs (6.5×106 cells/mL), after multiple perforations in 24 patients, and compared with a cell-free control group [17]. The author observed an improvement in the histological and arthroscopic scores (modified Shino and Wakitani scores) [16,58] during the 95 weeks of follow-up, which was better than that of the cell-free control group. As early as 4–10 weeks after cell transplantation, the defects were covered with soft tissue, which 28–95 weeks later, became a harder repair tissue and then, a cartilage-like tissue. Clinical examinations, assessed using the Hospital for Special Surgery Knee-rating scale, could not detect differences in the treatment procedure at the short-term follow-up, although histological and arthroscopic differences were observed. Wakitani concluded that MSCs are a potentially useful source for repairing tissues and the regenerated cartilage, probably, making the long-term follow-up results better and reducing the necessity for a subsequent total replacement [17]. This is the only paper, in this review, that used a control group.
In 2011, Wakitani et al. underlined the safety of the use of expanded MSCs (5×106 cells/mL) for OA and other cartilage defects at a long-term follow-up (11 years) of 41 patients [57]. Neither tumors nor infections were observed after MSC transplantation in these patients.
Partial and full-thickness articular cartilage defects
Kuroda et al. studied the effectiveness of autologous expanded MSCs (5×106 cells/mL) to repair a full-thickness articular cartilage defect in the weight-bearing medial femoral condyle of an athlete. After 7 months, the defect was filled with hyaline-like tissue (Safranin-O and Toluidine Blue staining), which was positive for collagen II (immunohistochemical analysis) and possessed a smooth surface that was assessed by arthroscopy (International Cartilage Research Society [ICRS] score). After 1 year, the clinical symptoms had improved, and MRI revealed focal chondral irregularities. The author concluded that the transplantation of autologous MSCs could promote the repair of large focal articular cartilage defects in young, active patients [32].
Wakitani, et al. extensively studied, over several years, the clinical regeneration of full-thickness articular cartilage defects, with the transplantation of autologous culture-expanded MSCs. In 2004, they observed that the repair of patella articular cartilage, treated with autologous-expanded MSCs (5.6×106 cells/mL) in 2 patients, after multiple perforations, was much faster than natural repair; arthroscopic and histological examinations (Toluidine Blue staining) revealed that the defect was covered with softer tissue after only 2 months and with cartilage-like tissue, after 1–2 years. Clinical symptoms (pain and walking ability) improved after 6 months and remained good for a long time (4–5 years) [33]. In another study conducted in 2007, Wakitani et al. also observed an improvement in the clinical symptoms of full-thickness articular cartilage defects of the patella-femoral joints in 3 patients. Autologous culture-expanded MSCs (5×106 cells/mL) were used, after multiple perforations in 1 patient, and straightforward implantation in the other 2. Clinical symptoms [International Knee Documentation Committee (IKDC) score] improved and remained satisfactory for the duration of the observation periods (27 months). Arthroscopic, histological (Hematoxylin and eosin and Toluidine Blue staining), and MRI evaluations showed that a cartilage-like tissue covered the defects, and the regenerated tissue was composed of a cartilaginous-like matrix [34].
Kasemkijwattana et al. described improvements clinically [knee injury and osteoarthritis outcome score (KOOS) and IKDC scores], and arthroscopically in large cartilage defects of the lateral femoral condyle of the knee, at 31 months follow-up in 2 young patients, after treatment with expanded MSCs [35], without reporting the cell concentration.
To summarize this first part of the review, a variability has been found with regard to the lesion size, the amount of delivered cells, the culture passages to obtain an adequate cell number to be implanted, the types of scaffolds, the number of patients, and the length of follow-up. Most of the authors reported information about the lesion size by expressing it as an area without providing precise data on lesion thickness, and one of the biggest cartilage defects repaired by expanded MSC transplantation was about 12 cm2 [33]; only Centeno et al. reported the lesion size as volume (4,020 and 4,668 mm3) and in 2011, Wakitani et al. did not specify the lesion dimension [57]. The amount of in vitro expanded MSCs, delivered into cartilage defects, varied from 1.3×107 cells [17] to 4.6×107 cells [52]. Thus, a comparison between studies is quite difficult due to the impossibility to normalize the delivered MSCs with the same lesion unit dimension (mm2 or mm3). However, according to reported and calculated seeding MSCs concentrations, it could be extrapolated that cell density ranged from 3.8×106 to 11.2×106 cells/mL with an average value of 5.8±2.3×106 cells/mL.
Regarding the number of culture passages to obtain the best amount of MSCs to be implanted, the most analyzed studies employed MSCs after 20–30 days of culture [17,50 –53,57] and in 2 studies, at the 3rd or 5th culture passage [52,53].
Besides a single case report study [53], which described injected expanded MSCs without scaffolds, all the others reported the use of collagenic [17,32 –35,57] or hyaluronic membranes [48] that served as a cell support with periosteum or synovium flaps [17,32 –35]. Such membranes are used to prevent MSCs from being lost inside the joint space, by acting as a biological chamber that protects and contains cells as they differentiate into chondrocytes, forming a healthy regenerative cartilage, and providing a suitable environment that synthesizes matrix macromolecules [17]. Since the chondrogenic potential of periosteum is thought to decrease with age, after 40 years [59], Wakitani et al. used synovium to cover the transplanted MSCs in the lesion site, thus suggesting that it has higher chondrogenic potential in a patient older than 40 years [34]. Finally, Centeno et al. added PL to the implanted constructs, because it contains GFs, which facilitate the growth and differentiation of MSCs [52,53].
Most articles described 1, 2, or 3 patients [52,53,32 –35], but 2 articles analyzed 24 and 41 patients [17,57], respectively, and the maximum follow-up was 11 years, if Wakitani's study about safety was considered; while when only trials that analyzed clinical outcomes were considered, the maximum follow-up was 5.8 years.
One-step procedure
The idea of transplanting the entire BM (BM in toto) or BM concentrate, by transferring the entire regenerative potential present in the BM environment to the lesion site, permits the cells to be processed directly in the operating room, without the need for a laboratory phase, thus allowing the transplantation to be performed in “one step” [60], therefore reducing the costs, risks and not requiring a GMP facility. Unlike two steps, the one-step arthroscopic technique is also used for the regeneration of osteochondral lesions [19,49,50], with clinical and histological results being consistent with those of previously published studies that present a series of patients with similar lesions treated by arthroscopic ACI.
Seven papers regarded the one-step procedure (Table 2): 1 paper focused on OA lesions [51]; 3, on partial and full-thickness articular cartilage defects [54 –56]; and the remaining 3, on ostechondral lesions [19,49,50].
PRF, platelet-rich fibrin gel; PRP, platelet rich plasma; FGRE, fast gradient recall echo; AOFAS, American Orthopaedic Foot and Ankle Society; MOCART, magnetic resonance observation of cartilage repair tissue; CRA, cartilage repair assessment.
Osteoarthritis
In 2006, Centeno et al. published a case report that described partial articular surface regeneration in a severely degenerated hip, using a one-step technique, 4–8 weeks after 2 autologous nucleated cell BM injections, in a patient. Three months later, an improvement was observed in traveling, recreation and standing tolerance, walking distance and sitting tolerance [51]. All these outcomes were evaluated using a modified VAS score, Functional Rating Index; lumbar range of motion was assessed using double inclinometry and the Coronal Fast Gradient Recall Echo sequence using identical 2nd preprocedure and postprocedure protocols.
Partial and full-thickness articular cartilage defects
Slynarski et al. combined periosteum with fresh BM in toto implantation as a source of MSCs for the treatment of a full-thickness cartilage defect in 14 patients. They concluded that the combination of these 2 sources of cells with chondrogenic potential may be considered an alternative for the treatment of cartilage defects. Pain improved after only 3 months and in 85.7% of cases, the IKDC and the modified Cincinnati grading scores significantly increased after 12 months. MRI showed such cartilage surface continuity that it could be considered normal or nearly normal. Moreover, they found a positive correlation between large-size defects and poor results [54].
In 2010, Pascarella et al. described a modified AMIC technique for the treatment of chondral lesions of the femoral condyle or patellar articular surface in 19 patients; they obtained healthy regenerative cartilage that could be compared with that achieved with ACI from a biological point of view. The AMIC technique consisted of bone perforations according to Pridie [61], and covering with a biological collagen patch enriched with BM in toto drawn through the perforations themselves. This one-step technique offered more advantages, because it combined the benefits of both the Pridie technique and the in situ proliferation of mesenchymal cells on a biological collagen membrane. The IKDC score, modified Ikeuchi score, and Lysholm knee Scale increased, and the MRI showed a reduction in the defect area, shape, filling, interface, and subchondral oedema in about half of the patients throughout the follow-up (24 months). The most important findings of this study was that a high amount of MSCs could be accumulated in the cartilage defect area by both perforations and the use of a membrane enriched by BM in toto [55].
Gigante A et al. performed a pilot study to analyze the histological quality of second-look biopsies taken from 5 patients who underwent a combination of microfractures and BM concentrate to treat isolated symptomatic articular cartilage lesions of medial femoral condyle of the knee. Clinical and histological scores (ICRS II) suggested that this procedure achieved a nearly normal (ICRS-cartilage repair assessment) appearance and a satisfactory repair tissue at 12 months follow-up. The repaired tissue (Safranin-O staining) was fibrocartilage in 3 cases, hyaline-like cartilage in 1 case, and a mixture of hyaline/fibrocartilage in 1 case. BM concentrate on a collagen scaffold may differentiate into chondrocytes and create fibrocartilage or hyaline extracellular matrix (ECM) in the lesion site [56].
Osteochondral lesions
In 2009, Giannini, et al. studied 48 patients, with focal osteochondral lesions of the talar dome treated with transplantation of BM-derived cell concentrate using a one-step technique, which was carried out using 2 different scaffolds: collagen powder (23 patients) or hyaluronic acid membrane (25 patients) enriched with platelet-rich fibrin (PRF) gel. They found that both patient groups exhibited a similar pattern of improvement at each follow-up. The clinical improvement and percentage of maximum possible improvement in the AOFAS score between the 2 groups were similar at each follow-up. In fact, the majority of patients returned to high-impact sports activity within 10–12 months after treatment, achieving an average AOFAS value of 91.4±7.9 at 24 months (collagen powder group: 89.8±9.8; hyaluronic acid membrane group: 92.8±5.3). The MRI showed newly formed tissue at the lesion site in all patients at 24 months. At 12 months, a second-look biopsy was performed in 5 patients (3 patients were asymptomatic, and 2 were symptomatic with hypertrophic regenerated tissue), who received collagen scaffold. Histological (Haematoxylin and eosin and Safranin-O stainings) and immunohistochemical (Collagen Type I and II) results confirmed the presence of new cartilaginous tissues with various degrees of tissue remodeling toward hyaline cartilage, with a complete integration between implant and native cartilage [19]. The results suggested that the one-step technique is an alternative for cartilage repair, and it improves functional scores.
In 2010, in another clinical study, Giannini S et al. studied the repair potential of the transplantation of BM concentrate, with a one-step technique, in 25 young patients with focal osteochondral monolateral lesions of the talar dome. Even if further research is required to regenerate a hyaline cartilage with biomechanical properties similar to normal hyaline articular cartilage, this technique produced highly satisfactory results over time (follow-up of 36 months). The results were excellent or good in more than 80% of cases and did not show any negative tendency over time. The radiographic, MRI [magnetic resonance observation of cartilage repair tissue (MOCART) scoring system], and histological (Safranin-O staining) findings showed, in most cases, a good restoration of the cartilaginous layer and a regenerated tissue in reorganization that approximated the characteristics of the original hyaline cartilage; the AOFAS score increased, and there was complete integration with the surrounding cartilage and a continuous and intact cartilage layer with evidence of collagen II (immunohistochemical analyses) and proteoglycans [49]. These 2 papers suggested that the one-step technique is an advance in osteochondral regeneration and offers good clinical outcomes and an integrated cartilage with similar histological characteristics to native adjacent tissue, with the addition of PRF as GFs supplementation.
Twenty patients with osteochondral lesions of the femoral condyle were analyzed by Buda et al., with BM concentrate with the supplement of PRF to provide GFs, during a follow-up of 24 months. The clinical IKDC and KOOS scores improved during the follow-up, with regeneration of subchondral bone (MRI MOCART score) and cartilage tissue rich in proteoglycans and type II collagen (Safranin-O and Haematoxilin and Eosin and immunoistochemical collagen Type II and I staining), thus concluding that this technique is a good treatment for osteochondral defects [50].
Regarding the one-step procedure, a combination of BM in toto and periosteum was used for the treatment of full-thickness cartilage defects, and showed that their application has a chondrogenic potential, which may be considered another option for the treatment of cartilage defects [54]. In a case report by Centeno et al., 2 sequential deliveries of nucleated BM cells were used to regenerate cartilage tissue in an osteoarthritic hip and, even if no conclusions can be drawn from 1 case report, these results may have implications for interventional pain management [51].
As in the two-step technique, in the one-step procedure as well, a variability was observed with regard to the lesion area, the scaffolds used, the number of enrolled patients, and the length of follow-up. The largest cartilage lesions found in these papers was about 6.8 cm2 [54], and most of the clinical trials employed collagen or hyaluronic acid as the delivery method for BM cells [19,49 –51,55,56]. Interestingly, Centeno et al. adopted the one-step technique combined with a biological stimulus, such as Platelet Rich Plasma, to treat OA cartilage lesion in the oldest patient [51]. The 2 studies by Giannini et al. and 1 study by Buda et al. also applied PRF to enrich the implantable construct of GFs such as platelet-derived growth factors AA, BB, and AB, thus transforming growth factor β1 and β2, platelet-derived epidermal growth factor, platelet-derived angiogenesis factor, insulin growth factor 1, and platelet factor 4, to enhance bone regeneration [62] in the osteochondral lesions.
Unlike the previous studies conducted on the two-step procedure, the one-step studies enrolled more patients, from 14 to 48 [19,49,50,54,55], and only 2 studies 1 or 5 patients [51,56], with a maximum follow-up of 3 years.
An additional important variable to be taken into account for the clinical success of the 2 techniques is the donor age. In a healthy population, the decreased regenerative potential of our body is considered as depending on the loss of MSC potentiality and the changes observed in the regulation of the microenvironment differentiation pathways occurring during aging [63]. In a previous review conducted by the present authors, contrasting papers were found on the in vitro behavior of MSCs during aging [63], and, therefore, MSC donor age may be considered critical for their clinical applications; so, it is very important to assess the efficacy and the potential of MSC when isolated from elderly patients. In all the papers examined in this review, the age range was between 18 and 70 years old, but most articles recruited young or middle-aged subjects [19,49, 51,52,32 –35,54 –57]; whereas only 2 studies enrolled older patients [17,51]. In 1 case, the age of the patients was not described [50].
Finally, the safety, linked to the use of MSCs in clinical practice, is a very important aspect to be evaluated, but information in humans is limited [64,65] and recent controversies about the stability of hMSCs are highlighted. The safety of hMSCs is frequently assessed with in vitro studies to investigate the potential susceptibility of these cells to malignant transformation, but conflicting results are reported: some authors observed a spontaneous transformation of MSCs, especially in long-term cultures [66 –68]; whereas others found that MSCs maintain their characteristics without developing chromosomal aberrations [69,70]. Studies conducted with animals, most of them performed in syngenic or immunodeficient small-sized animals, also found the same contradictory results [71 –75]. By considering the current review, on clinical studies, only Wakitani et al. tested the safety of autologous MSCs in humans, observing no malignant transformations or infections [57].
Conclusions
From this review, some considerations can be made based on the 2 techniques analyzed. First of all, taking into consideration all the papers analyzed, the largest cartilage defect repaired with the two-step technique is about twice the size of that observed in the one-step approach, although some authors do not indicate the lesion size. Second, all the studies conducted on the two-step approach, despite having a lower number of patients, have a mean follow-up of about 2- or 4-fold more than those conducted on the one-step technique. Third, the number of implanted cells is not specified in the one-step papers, and there is a lack of uniformity in the amount of expanded MSCs used in the two-step technique. Regarding lesions, OA defects are treated mainly by the two-steps technique, because it seems much more able to improve the MSCs survival, limiting the negative effects of the OA joint environment. In fact, in order to limit the damaging effect of the inflammatory microenvironment (IL-1β, IL-6, and TNF-α), it has been proposed that mature constructs should be implanted, because the presence of an ECM may protect cells from the chemical assault [76,77]. On the contrary, osteochondral defects are treated with the one-step method. However, there is agreement about delivering MSCs in both procedures by using a scaffold (collagen or hyaluronic acid) that can carry and retain cells in the lesion site and by covering the lesion site with biological patches to also protect the transplant from mechanical load. Finally, no differences were observed between the mean age of all patients treated with the one-step and two-step techniques (39.1 and 39.2 mean age for one-step and two-step techniques, respectively).
In conclusion, from a laboratory point of view, there is a lack of standardization of the adopted methodology, both for expanded MSCs and for the one-step procedures, with different amounts of cells and culture conditions. Conversely, from a clinical point of view, there are different follow-up times; different types of lesions, regarding etiology, dimension, and localization; and different kinds of patients, regarding age, clinical background, and associated pathologies. Thus, it follows that all these variables affect the strength and reliability of experimental results. Many authors suggested that the size of the lesion as well as the osteoarthitic nature of the lesion are the main factors which are correlated with poor clinical outcomes.
Despite these considerations, clinical outcomes of MSC transplantation procedures seem to be highly satisfactory. Although further research is required to regenerate a hyaline cartilage that is indistinguishable from native articular cartilage, the techniques proposed permitted highly satisfactory results over time. Although longer follow-up and randomized controlled clinical trials are needed to confirm the validity of the cartilage repair over time, the one-step technique represents an advance in cartilage and osteochondral defect regeneration, which offers a rapid process, high clinical scores, and a tissue repair that is similar to the healthy tissue and without the major disadvantages linked to other techniques.
Footnotes
Acknowledgments
This article was partially supported by Rizzoli Orthopaedic Institute-IOR as well as by the “pathogenesis and molecular targets in degenerative musculoskeletal diseases” project FIRB no. RBAP10KCNS-2010.
Author Disclosure Statement
The author confirms that no conflicts of interest are associated with this publication, and no competing financial interests exist that could have influenced its outcome.