The use of mesenchymal stem cells for cartilage repair and regeneration: a systematic review

Background The management of articular cartilage defects presents many clinical challenges due to its avascular, aneural and alymphatic nature. Bone marrow stimulation techniques, such as microfracture, are the most frequently used method in clinical practice however the resulting mixed fibrocartilage tissue which is inferior to native hyaline cartilage. Other methods have shown promise but are far from perfect. There is an unmet need and growing interest in regenerative medicine and tissue engineering to improve the outcome for patients requiring cartilage repair. Many published reviews on cartilage repair only list human clinical trials, underestimating the wealth of basic sciences and animal studies that are precursors to future research. We therefore set out to perform a systematic review of the literature to assess the translation of stem cell therapy to explore what research had been carried out at each of the stages of translation from bench-top (in vitro), animal (pre-clinical) and human studies (clinical) and assemble an evidence-based cascade for the responsible introduction of stem cell therapy for cartilage defects. Main body of abstract This review was conducted in accordance to PRISMA guidelines using CINHAL, MEDLINE, EMBASE, Scopus and Web of Knowledge databases from 1st January 1900 to 30th June 2015. In total, there were 2880 studies identified of which 252 studies were included for analysis (100 articles for in vitro studies, 111 studies for animal studies; and 31 studies for human studies). There was a huge variance in cell source in pre-clinical studies both of terms of animal used, location of harvest (fat, marrow, blood or synovium) and allogeneicity. The use of scaffolds, growth factors, number of cell passages and number of cells used was hugely heterogeneous. Short conclusions This review offers a comprehensive assessment of the evidence behind the translation of basic science to the clinical practice of cartilage repair. It has revealed a lack of connectivity between the in vitro, pre-clinical and human data and a patchwork quilt of synergistic evidence. Drivers for progress in this space are largely driven by patient demand, surgeon inquisition and a regulatory framework that is learning at the same pace as new developments take place.

Bone marrow stimulation techniques, such as microfracture, are the most frequently used method in clinical practice for treating small symptomatic lesions of the articular cartilage [6][7][8]. However, the resulting tissue has shown to be a mixed fibrocartilage tissue [20][21][22] with varying amounts of type II collagen [8,21,23,24] and inferior to native hyaline cartilage. Fibrocartilage is vulnerable to shear stresses and prone to breaking down over time [20]. Subchondral osseous overgrowth has also been reported after microfracture [25,26]. Osteochondral grafts can lead to donor site morbidity and healing seams at the recipient site [27,28]. Autologous chondrocyte implantation (ACI) [9,10] and its later evolution, matrix-induced autologous chondrocyte implantation (MACI), offered great promise with 80% of patients showing good or excellent results at 10 years [29] but at best results in hyaline-like repair and has experienced complications such as graft failure, periosteal hypertrophy and delamination [30,31]. In addition, it has also been reported that cells may lose their phenotype during expansion [32,33].
There is therefore a growing interest in regenerative medicine, which can broadly be thought of as two main types: cell therapy, where cells are injected directly into the blood or into tissues, and tissue engineering, where cell-scaffold combinations are used to repair or regenerate tissues.
Stem cells are cells that have the ability to divide and develop into many different cell types in the body and can be categorised as pluripotent and multipotent. Pluripotent stem cells are often harvested from embryonic sources and can develop into any type of cell in the body whereas multipotent stem cells are generally taken from adults and can divide and develop into a more limited range of cell types. When stem cells divide, the new cells can either remain stem cells or develop into a new type of cell with a more specific function (Table 1).
Mesenchymal stem cells (MSCs) are a form of multipotent cells that may offer an alternative to cartilage repair techniques not hampered by availability and donor site morbidity.
The introduction of stem cell therapies into clinical practice however is a form of translational research, which as per any "bench-to-bedside" pathway now has enormous governance issues [34,35] and is highly regulatory across four phases (Table 2) and by the Tissues and Cells Directive (2004/23/EC) https://www.hta. gov.uk/policies/eu-tissue-and-cells-directives.
Many published reviews on cartilage repair only list human clinical trials [13,[36][37][38][39][40][41][42][43][44][45][46], underestimating the wealth of basic sciences and animal studies that are precursors to future research and may be relevant in clinical practice further down the line. In addition, true translation would imply that all of the clinical studies would have supporting pre-clinical data.
We therefore set out to perform a systematic review of the literature to assess the translation of stem cell therapy to explore what research had been carried out at each of the stages of translation from bench-top (in vitro), animal (pre-clinical), and human studies (clinical) and assemble an evidence-based cascade for the responsible introduction of stem cell therapy for cartilage defects. In particular, we wanted to focus on the key burning questions pertaining to cartilage repair such as cell source, dosage (how many cells should be used), requirement for scaffolds and the role for extrinsic growth factors.

Search methodology
This review was conducted in accordance to PRISMA guidelines [47] using CINHAL, MEDLINE, EMBASE, Scopus and Web of Knowledge databases from 1st January 1900 to 30th June 2015.
The keywords used in the selection were "("mesenchy- All review and non-English studies were excluded. For analysis, only original research studies were included. Any duplicates were excluded. Initially, KM and JS independently screened studies' title and abstract. Those • They are unspecialized ("blank slates" that can become specific types of cells).
• They can develop into specialized cell types (cells that do specific work in the body).
• They are capable of surviving over long periods and divide to make additional stem cells. Phase II: Uncontrolled Efficacy Studies. The drug or treatment is given to a larger group of people to see if it is effective and to further evaluate its safety.
Phase III: Randomised Clinical Trials. The drug or treatment is given to large groups of people to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the drug or treatment to be used safely.
Phase IV: Post-Market Surveillance. Studies are done after the drug or treatment has been marketed to gather information on the drug's effect in various populations and any side effects associated with long-term use.
included had the full text reviewed. Any disparities were discussed with the senior author (AJG). The references of eligible studies were also searched and included where relevant. Unpublished trial databases (e.g. ClinicalTrials.gov) were reviewed as the grey literature using popular search engines, including Google. The keywords used for registered clinical trials in clinical trial databases were "stem cells", "cartilage" and "orthopaedics".
Eligible studies were drafted into tables tabulating the key data.

Results
The initial search identified 2880 study articles, of which 239 were included for analysis. The PRISMA flow diagram is shown in Fig. 1.

In vitro studies
MSC source A list of cell sources used in the in vitro studies is shown in Table 3. The commonest being human MSCs (66%) followed by rabbit MSCs (15%). The majority of the studies used bone marrow-derived MSCs (63%) followed by adipose tissue (33%). Two studies used commercial cell lines [48,49].
Scaffold Within the in vitro studies, 26 different types of natural scaffold and 9 types of synthetic scaffolds were identified with a further 18 different types of hybrids, the most popular being a fibrin-polyurethane scaffold (Table 4).

Growth factors
The commonest used growth factors were TGF-β and the bone morphogenetic protein (BMP) family. A list of growth factors used can be seen in Table 5.
Cell seeding and passage There was wide heterogeneity in cell seeding density and there appeared to be no standard form of measurement. Li et al. [50] examined three different seeding densities: 2, 5 and 10 × 10 6 cells/ scaffold, and found that scaffolds seeded with 5 × 10 6 cells per scaffold induced the highest chondrogenesis; however, other groups [51][52][53] found that a higher seeding density results in better chondrogenesis. Apart from 26 studies which did not state cell passage number, most studies used MSC of an early passage, anything between uncultured fresh (passage zero (P0) and five times passaged cells (P5). One study used cells of P6 [54], and another study used cells between P4 and P7 [48]. No relationship was apparent between chondrogenesis and number of passages.
Length of study The length of each in vitro study can be seen in Table 6. The majority of studies were shortterm models; 27 studies (25%) ended between 1 and 2 weeks, 35 studies (33%) ended between 2 and 3 weeks and 15 studies (14%) ended between 3 and 4 weeks.
Method of assessment A range of techniques was used to assess chondrogenesis within the in vitro studies. These techniques consisted of histology, immunohistochemistry, qPCR, biochemical analysis, imagery and mechanical testing. The techniques used are summarised in Table 7.
Animal studies (pre-clinical) One hundred eleven animal studies were included of which 109 were controlled laboratory studies, one was a  [49] and one was a longitudinal case study on a race horse [55]. The commonest animal studied with 59 studies was rabbit (53%). The different species of animals studied is shown in Table 8.
Defect The size of the defect varied from 2 to 25 mm 2 in the smaller animals and from 1 to 64 mm 2 in the larger animals. All but two studies [56,57] used the knee for defect creation.
Cell culture, dose and delivery There was much variation in the number of cells implanted and the number of cell passages from 3-10 or more [79,86].
The number of cells varied from 4 × 10 3 -1 × 10 10 . The majority of studies used between 10 6 and 10 8 cells. Some did not specify the number of cells implanted. Two studies suggested that improved chondrogenesis occurs with a higher implanted cell number [75,87], although others suggested that the high cell numbers increase the risk of synovitis [75] and synovial proliferation [88].
Growth factors Thirty-two studies (29%) assessed the effect of growth factors on MSC chondrogenesis. Seventeen out of 38 (44%) used TGF-β1/3 (Table 10), the majority of which show a positive effect on chondrogenesis.

Human studies (clinical)
Thirty-one published studies by 15 different groups looked at clinical applications of MSCs. One used allogenic stem cells [103] and the rest autologous stem cells.
The types of studies can be seen in Tables 13 and 14. There were 52 unpublished clinical trials, majority of which are early phase studies (I-II; 63%) and only 5 trials were phase II/III. Table 15 shows a summary of these clinical trials.
In the cell therapy studies, the cells were suspended with a variety of different co-stimulators, including hydroxyapatite (HA) [106,119,121,123], platelet rich plasma (PRP) [106,114] and platelet lysate [104]. Some studies also administered multiple injections of stem cells [119,121] and/or further injection of HA [115,119,121,123], PRP [106,114] or nucleated cells [104] following a stem cell injection.
The most frequently used scaffolds were type I collagen of porcine or bovine origin [113,118,122,124,126,129], followed by ascorbic acid sheet [120,123] and platelet-rich fibrin glue mixture [108,125].
Adverse effects None of the studies reported any severe adverse effects related to the MSC treatment. Two group reported minor adverse events including mild pain and effusion after the injections, which persisted for no more than 7 days [103,114].

Conclusions
There is a growing fascination with the role of mesenchymal stem cells in cartilage repair.
Cartilage repair has evolved from marrow stimulation techniques through to chondrocyte transplant and now stem cells at rapid pace. An ideal translational pipeline would demonstrate how in vitro data was used to inform a pre-clinical model, which would later form a phase I/IIa first-in-man study and subsequently a phase III clinical trial. This would of course be the safe and responsible method by which novel therapies are brought to the market.
This systematic review is the first of its kind to explore the full spectrum of evidence from in vitro studies, through animal studies to human clinical trials, and yet, we found little evidence of connectivity between in vitro, animal and then human work. In fact, we did not find a single group that had carried out and reported studies in all three categories.
Indeed, even from groups, which showed a seemingly hierarchical approach to translation, discrepancies became apparent. For example, Saw et al. from Korea used a pre-clinical goat model to repair cartilage defects using HA plus bone marrow-derived cells [150] and then moved into a first-in-man study, but in doing so, elected to change from bone marrow aspirate to peripheral blood and justified this change because it was easier to harvest peripheral blood than marrow [151].
There are several sources of cells that have been used in cartilage repair including bone marrow, peripheral blood, synovium, adipose tissue and umbilicus (Table 14) without any clear evidence of superiority of one over the other.

One stage vs. two stages
As two stage procedures involving cell culture are expensive and cumbersome, there is an increasing push towards a single stage stem cell treatment. In this situation there is some supportive pre-clinical data [91,95,98,[152][153][154], but there does not appear to be a pre-clinical study that directly compares bone marrow concentrates against cultured MSCs.
Several groups have reported the use of bone marrow concentrates in clinical practice [116,117,127,128,[130][131][132], in which the buffy coat is used containing the nucleated cells, of which a few will be stem cells.
Briefly, the patient has approximately 60 mL of bone marrow harvested from the iliac crest which is then spun down in a cell centrifuge (SmartPrep, Harvest Technologies Corp., USA, or IOR-G1, Novagenit, Mezzolombardo, TN, Italy) to provide 6 mL of concentrate containing nucleated cells. A small amount of the nucleated cells are then placed onto a hyaluronic acid membrane (Hyalofast, Fidia Advanced Biopolymers, Italy) or collagen membrane (IOR-G1, Novagenit, Mezzolombardo,    [129,133] Cell delivery
There has also been a further step taken to avoid bone marrow harvest in which peripheral blood has been used in knee chondral lesions. In an RCT, arthroscopic subchondral drilling was followed by postoperative intraarticular injections of hyaluronic acid (HA) with and without peripheral blood stem cells (PBSC). Fifty patients were studied and randomised 1 week after surgery to receive either 8 injections of HA or 8 injections of HA plus PBSC. Those that underwent PBSC received stimulation with filgrastim, which contains recombinant human granulocyte colony-stimulating factor prior to harvest [106,151]. At 18 month follow-up, they reported no adverse effects and improved MRI findings in the PBSC group compared to HA alone, took biopsies of 16 of the 25 patients in each group and claimed better tissue morphology in the PBSC group, as graded by the International Cartilage Repair Society Visual Assessment Scale II. Interestingly, however, the same group's preclinical used bone marrow aspirates and not peripheral blood [150].

Autologous vs. allogenic
There is an increasing interest in allogenic cells to avoid donor site morbidity and to reduce cost. The pre-clinical data with regards to allogenic cells is conflicting. One group showed promising results of allogenic MSCs in a rabbit model when compared to autologous cells, although numbers were small [160,161]. Another group compared autologous chondroprogenitor cells and allogenic chondroprogenitor cells against controls in an equine model and reported inferior repair in the allogenic cell group [23]. Despite conflicting pre-clinical data, human studies using allogenic cells began in Korea in 2009. A phase I/IIa study to assess safety and efficacy of a combination of human umbilical cord bloodderived mesenchymal stem cells and sodium hyaluronate (CARTISTEM ® (MEDIPOST Co., Ltd., Korea)) was performed in knee chondral defects (NCT01041001). A parallel phase 3, open-label, multi-centre RCT comparing CARTISTEM ® and microfracture in knee chondral defects was carried out in Korea and the USA (NCT01733186). Results are still pending.
Another area of huge controversy is the actual dose of cells that should be used. In vitro between 50,000 cells/ mL and 100 billion cells/ml have been studied. In preclinical animal studies, this ranged from 1000 to 1 billion cells/mL, and in human studies, the reported range has been 1.2 million cells/mL-24 million cells/mL.
It remains unclear what the most appropriate cell dose should be, with some groups reporting that a higher cell number leads to a better repair [52,71,87,95,[162][163][164], but Zhao et al. [99] highlighted the limitation to cell saturation and survival, and thus, there may be a top limit to cell number that can be used to aid repair.
A multitude of methods for cell delivery have also been adopted, from direct joint injection or embedded in a plethora of scaffolds, such as type I collagen gels of porcine or bovine origin, ascorbic acid sheets or fibrin glues (Table 14).
In vitro and in pre-clinical studies, a plethora of growth factors have been studied including TGF-β1 and TGF-β2 and BMP-7 but none of these have been included in human clinical trials (Table 5).
It is clear that the relationship between cell passage, cell dose, the use of scaffolds and growth factors and the efficacy of MSC treatment is still to be established.

Future
There is no question that the field of cartilage repair accelerates at rapid pace, and it is clear that the single stage procedures are likely to win over two stage procedures to save costs and reduce the burden on both provider and the patient. The reduction of donor site morbidity is a further driver helping direct progress.
The concept of cell banks of allogenic cells clearly meets all of the above criteria, but the lack of good supporting pre-clinical and long-term safety and efficacy data does little to pacify potential pitfalls of this direction. The fact that the phase 3 RCT of allogenic umbilical stem cells was allowed to be registered (NCT01041001) before the same group registered their phase I/IIa safety study (NCT01733186) intimates that sometimes clinical pace exceeds that of the regulators to lay down new ground.
Tools are likely to be introduced to the operating theatre that might improve the efficacy of treatment, such as fluorescence-activated cell sorting (FACS) machines which can isolate MSCs from the buffy coat of bone marrow aspirate by their cell surface markers. At present, this technology is expensive and complicated and ways to reduce cost and make the process simple are required before they could enter the operating theatre.
Induced pluripotent stem cells (iPSCs) are adult somatic cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells [165].
These cells show unlimited self-renewal, and some in vitro studies have shown chondrogenic differentiation by iPSCs from human chondrocytes biopsied from osteoarthritic knees [166] and cartilage formation from human neural stem cells [167]. However, this work is at a very early stage, and aside from the ethical considerations, much research into control of cell phenotype and cell fate to alleviate concerns for cancer risk are required before this technology is ready to move into the preclinical and clinical realms.
In conclusion, this review is a comprehensive assessment of the evidence base to date behind the translation of basic science to the clinical practice of cartilage repair. We have revealed a lack of connectivity between the in vitro, pre-clinical and human data and a patchwork quilt of synergistic evidence. It appears that the drivers for progress in this space are largely driven by patient demand, surgeon inquisition, and a regulatory framework that is learning at the same pace as new developments take place. We strongly recommend funding body commission studies that have a clear translational purpose in order to drive the science towards patient benefit.

Funding
There was no external funding for this work.

Availability of data and materials Not applicable
Authors' contributions All authors were involved in the conception and design of the study or acquisition of the data or analysis and interpretation of the data and contributed to drafting the article or revising it critically for important intellectual content. All authors read and approved the final manuscript.

Competing interests
The authors declare that they have no competing interests.

Consent for publication Not applicable
Ethics approval and consent to participate Not applicable Author details 1 Institute of Orthopaedics and Musculoskeletal Science, Royal National Orthopaedic Hospital (RNOH), Brockley Hill Stanmore, London HA7 4LP, UK.