Adipose Tissue-Derived Mesenchymal Stem Cells (AD-MSC) Versus Platelet-Rich Plasma (PRP) in the Arthroscopic Treatment of Chondral Defects of The Knee: What’s The Best? A Histological and Clinical Evaluation

Adipose Tissue-Derived Mesenchymal Stem Cells (AD-MSC


Introduction
Articular cartilage is a thin viscoelastic specialized tissue composed of chondrocytes dispersed within an extracellular matrix. Overall cartilage homeostasis is the product of a complex interaction between growth factors, joint mechanics, hormones and aging. [1] Native articular cartilage experiences and endures a myriad of mechanical forces, such as compression, tension, shear and hydrostatic pressure. [2,3] Chondral defects are commonly encountered in orthopedic practice since articular cartilage has a poor self-regeneration ability and focal articular defects occur in up to two-thirds of patients experiencing knee arthroscopy, though often asymptomatic. [4] Anyway, symptomatic cartilage lesions can significantly limit the common activities of daily living, with mild to severe pain or discomfort, so this situation can be truly disabling especially in healthy young patients. For this reason, different surgical options have been proposed for the management of isolated chondral lesions, such as microfracture, nanofracture, abrasive chondroplasty, and spongialisation. [5][6][7][8] All these techniques aspire to reduce pain, restore articular function and stimulate cartilage regeneration. The limited biomechanical properties of spontaneously formed repair cartilage contribute to its functional incompetence and deterioration; clinical results are only temporary and satisfying for low-grade cartilage defects. [9] To overcome the shortcomings of these arthroscopic stimulating techniques, the concomitant support of biologic alternatives such as growth factors or multipotent cells might potentiate and ameliorate the regenerative response [10].
Platelet-Rich Plasma (PRP) is an important source of growth factors and it has been widely used since the 1970s as the ideal solution for cartilage defects, due to the attitude to promote cartilage healing, decrease pain and improve clinical function. [11] Its effect is still temporary (being estimated to last up to 1 year) and it is still debated if PRP has a chondroprotective effect on the progression of the cartilage defect and joint degeneration. A modern approach in the regenerative area starts from the capability of mesenchymal stem cells (MSCs) to activate the healing processes through an immunomodulatory and paracrine mechanism. [12] The paracrine activity is responsible for their anti-inflammatory, anti-apoptotic, anti-fibrotic, angiogenic and mitogenic properties. [13] Adiposetissue derived mesenchymal stem cells (AD-MSCs), located in the stromal vascular fraction of subcutaneous adipose tissue promote tissue regeneration by secreting growth factors and cytokines and are an optimal source, given their large availability. [14,15] As stated above, the potential benefits of bone marrow stimulation techniques could be amplified by the association with other regenerative techniques, such as PRP and AD-MSCs. Based on these premises, we defined and conducted this study to evaluate the histological and clinical effects of PRP and AD-MSCs in the arthroscopic treatment of isolated monocompartmental cartilage defects of the knee joint.

Materials and Methods
This is a prospective randomized controlled study conducted at a single center including 46 patients (mean age: 52.6 years; range 38-73 years). Eligible patients included adults affected by an arthroscopically verified monocompartmental chondral defect (< 2 cm 2 ) classified as grade IV according to Outerbridge classification, located on the femoral condyle or tibial plate. Exclusion criteria are listed in Table 1. It was not possible to blind the patients as to what treatment they would have received, given that one treatment arm required a supplementary surgical gesture (a mini-liposuction for the harvesting of AD-MSCs). The study was consistent with ethical principles for medical research ratified by the World Medical Association (WMA) Declaration of Helsinki, having been approved by the Internal Review Board. All patients were carefully informed about the modality and purpose of the study, gave their approval, and subscribed to a specific informed consent form. Before treatment, baseline demographic data was collected.  All patients, preliminary evaluated with knee MRI [16] and clinical examination, underwent a diagnostic arthroscopy to confirm an isolated chondral lesion (Outerbridge grade IV). All the surgical procedures (diagnostic and therapeutic ones) were performed by the same skilled surgeon (senior author). The chondral defect underwent arthroscopic debridement with an arthroscopic abrader to form a stable edge of healthy cartilage and a bone marrow stimulation technique (microfracture). After that an autologous PRP injection was performed in all patients: 8 mL of venous blood was taken from each patient and centrifugated to separate the blood into the plasma, the buffy coat, and residual red blood cells and collect the desired quantity of PRP, which was then injected into the affected knee. At this point, patients were randomly assigned to one of the two groups (allocation ratio 1:1) by the only member of the staff with access to the allocation spreadsheet. In this way, 23 patients (group A) experienced miniabdominal liposuction to harvest the AD-MSCs which were then injected into the affected knee. The other 23 patients (group B) didn't receive any further treatment. Group characteristics at inclusion are shown in Table 2.   Patients were prospectively evaluated basally before surgery and at 1, 6, 12 and 24 months of follow-up using OKS and IKDC for general health status, and VAS for pain evaluation. The survey of all clinical data was performed by a member of the staff not involved in the surgical procedure.

Sample Size and Statistical Analysis
We considered this trial as a pilot study: we are aware that pilot studies should be undertaken before evaluation in a full randomized controlled trial, according to published guidelines. Sample size calculation was not possible since there is insufficient data published to date on the effects of PRP and AD-MSCson knee chondral defects. On this basis, we included 46 participants with 23 patients allocated to each group. [17] Demographic and clinical characteristics have been presented as means and Standard Deviations (SD) for data with anormal distribution and median and interquartile range for non-parametric data. P-values of <0.05 were considered statistically significant and they were inter preted based on Bonferroni correction. All statistical analyses were performed using MedCalc ® version 13.3.1.

Results
OKS, IKDC and VAS data were collected for all patients at 1,6, 12 and 24 months of follow-up. Mean IKDC scores for group A and group B at baseline, at 12 and 24 months (final follow-up) are shown in Figure 1. No significant statistical differences in mean IKDC scores were detected between the two cohorts. The mean IKDC score at 24-months follow-up in group A was 72.4±1.8 compared to 70.3±1.5 in group B. The increase in IKDC score from baseline to follow-up was significant for both groups. The OKS profiles with mean scores before surgery, at 12 and 24 months-follow-up for group A and group B are shown in Figure  2.There were no differences between the two groups in any of the OKS data collection, with a progressive improvement at 12 months  In both groups we appreciated a similar progression from baseline in VAS (Figure 3). VAS score dropped from 6.4±1.33 before treatment to 3.32±1.55 at 12 months follow-up and to 4.0±1.6 at 24 months follow-up for group A and from 6.12±1.27 to 3.12±2.10 for group B at 12 months and to 4.8±1.6 at 24 months.

Figure 3:
VAS score evolution over time in group A and Group B -We note a comparable reduction of pain intensity for both groups from one month to 12 months after surgery with a rebound effect of pain perception at 24 months followup.
8 patients (5 for group A and 3 for group B) experienced adverse events (swelling and pain with longer recovery time). They were treated with oral nonsteroidal anti-inflammatory drugs, ice and rest, with no functional limitations, once passed the acute phase (lasting a few days). We performed a second-look knee arthroscopy (3 patients for group A and 3 patients for group B) one year after the regenerative/arthroscopic treatment in 6 patients eligible for knee corrective osteotomy (high tibial osteotomy in genu varum): smooth hyaline cartilage was appreciated in all patients, thus justifying the good clinical outcomes. Specifically, a thick smooth hyaline-like cartilage with normal mineralization and predominantly viable cell population (comparable to native articular cartilage) was detectable in group A ( Figure 4) whereas a smooth resurfacing fibrocartilage with predominantly viable cell population and abnormal cartilage mineralization was present in group B. Macroscopic and microscopic histological evaluation of biopsy samples referred to ICRS visual histological assessment scale; all data are reported in Table 4. Surface  3  3  3  3  3  3   Matrix  3  3  3  2  1  2   Cell distribution  2  3  2  1  2  2   Cell populationviability  3  3  3  1  1  1   Subchondral bone  3  2  2  2  2  2   Cartilagemineralization  3  3 3 0 0 0

Discussion
The results of this study focus on the clinical and histological evolution in the treatment of knee chondral defects. No significant statistical difference emerged from data collected in IKDC, OKS, and VAS. A similar progression is detectable in the 2 cohorts since a progressive improvement is present from baseline to 1 year and subsequent deterioration of clinical performance in both groups (though the clinical parameters at 2 years were better than pre-operative situation). The most interesting aspect of this study (though the evident statistical limits), to our opinion, concerns the histologic scope. The synergic effect of two regenerative procedures, such as AD-MSCs and PRP (in association with bone marrow stimulation such as microfracture) can lead to a more performing neo-cartilage, with predominantly viable cell population and normal mineralization (thus mimicking the native articular cartilage). Different surgical Volume 7; Issue 09 J Orthop Ther, an open access journal ISSN: 2575-8241 techniques have been developed and refined over the last years to stimulate a spontaneous repair reaction in cartilage defects but none of them has been able to restore the desired hyaline cartilage. [18][19][20] For this reason, these treatments are usually unsatisfactory in the long term. Knee microfracture (a bone marrow stimulation technique) is one of the most popular solutions in orthopedic practice, since this technique is cheap, doesn't require specific skills, and doesn't hinder potential future surgical treatments.
Cartilage deterioration is characterized by a mechanical basis, due to the ultrastructural damage, but also by an inflammatory process, as a consequence of pro-inflammatory cytokines activation, such as tumor necrosis factor α (TNFα), Interleukins (IL), aggrecan, and metalloproteinases. Regenerative options investigated and developed over the last decades have focused on this inflammatory aspect, aiming to defuse these catabolic factors and promote a regenerative path. Based on these strategies, PRP and MSCs can be ideal candidates for this teamwork and can play a key role.
PRP is a safe non-surgical treatment for cartilage disease and in consideration of its potential role in tissue healing has been widely investigated both in preclinical and clinical studies. Progressive implementation of the scientific production on this focus has been found in the last 15 years. [21] One of the actual limitations is the lack of standardization in PRP preparation and application, with no consensus on a definitive protocol. It is known that PRP preparation has a direct impact on the final composition but the relative implications are not clarified. Different classification systems for PRP have been proposed [22][23][24][25] but none of these has been approved and validated by an international consensus. Lacko et al. [26] investigated the metabolic effect of intra-articular applications of PRP in cartilage disease: according to their findings, a decrease in pro-inflammatory biomarkers associated with an increase in specific anti-inflammatory and pro-anabolic biomarkers was detectable 3 months after PRP treatment. A recent review by Ip et al. [27], analyzing 1093 patients among 23 trials, focused on the safety, efficacy, and indications of intra-articular injections of PRP and MSCs in knee osteoarthritis. According to this study, the effectiveness of PRP injection in knee osteoarthritis is undoubtedly positive, with better clinical outcomes in patients with low-grade articular consumption (Kellgren-Lawrence grade I-II) and better results with increased dosages of PRP by repetitive injections. No significant adverse effects have been appreciated (the most common adverse reaction was a mild local self-limiting tenderness at the site of injection).
MSC plays an anti-inflammatory role in a different pattern by inhibiting the maturation of immune cells (monocytes and lymphocytes), suppressing natural killer cells, and preventing cell apoptosis and cartilage damage. MSC, acting in a paracrine way, can stimulate chondral differentiation and regeneration of hyaline joint cartilage (with similar contents of type II collagen and aggrecan if compared to native cartilage). The large availability (from a wide array of tissues, including adipose tissue), the absence of significant adverse effects, and the readiness to use of MSCs make interesting their use in the management of cartilage diseases. The most collateral morbidity effects were bruising and local discomfort after liposuction and injection procedures. Many studies in the last decades have investigated the therapeutic potential of PRP and AD-MSCs in the management of knee cartilage defects. The results of this pilot study indicate that both modalities are safe and effective to modify the natural history of disease progression and enhance overall function. In both groups, a significant reduction of pain in association with enhanced functional abilities has been appreciated. In light of the data collection of our study, we confirm that age is a key element in the regenerative ability of cartilage disease: better results have been found in younger patients (< 50 years). Another important element is BMI since it is largely responsible for stress forces loading on knee cartilage: overweight subjects unavoidably show worse outcomes. Undoubtedly the biological potential of PRP and AD-MSC is related to individual variability among patients, needing further in-depth research. There are some limitations in this study; first of all, the limited number of patients in both groups. A larger-scale study would be necessary (especially for an in-depth histologic evaluation) for clinical application. Second, the followup should extend to assess clinical outcomes in the long term. Third, we cannot be certain that multiple PRP/AD-MSC injections are more effective. For all these reasons, we consider this trial a Volume 7; Issue 09 J Orthop Ther, an open access journal ISSN: 2575-8241 pilot study, preliminary for broader and thorough research.

Conclusions
On the clinical side, it is not yet incontrovertible if the association "PRP and MSCs" could improve the outcomes of bone marrow stimulation techniques. Anyway, the histological results of this trial address the light to the regeneration of higherperformance cartilage, with a form closely similar to native tissue. Additional research needs to be done and undoubtedly a great effort is required at both the basic and clinical research fronts.
This study represents a preliminary step to get evidence on cartilage defects treatment on which orthopedic surgeons could make important treatment selection decisions.