Introduction

Cartilage injuries are said to frequently lead to incomplete defect-regeneration due to the limited healing properties of hyaline cartilage (Gomoll AH, Minas T. 2014). As a pre-arthrotic deformity, this failure of restoration often results in progressive degeneration of the affected joint and a continous function impairment (Shapiro F et al.1993, Steinwachs MR et al 2008). Arthroscopic one step microfracture is the most common technique for cartilage repairs worldwide (Steadman JR et al. 2002, Kreuz PC et al. 2006, Mithoefer K et al. 2009). This method induces the formation of a repair tissue yet carries insufficient biomechanical features as well as an inadequate long-term weight-bearing capacity (Kreuz PC et al. 2006, Mithoefer K et al. 2009). Firstly, more extended injury of the subchondral bone caused by the use of V-shaped top of microfracture instruments is considered to be causative. Secondly, the shallow depth of perforation (3 mm) achieved by these devices only allows very restricted access to the subchondral pool of stem cells with poorly developed cell differentiation characteristics (Chen H et al. 2011). This restriction becomes very apparent with the hypertrophic formation of bone within the defective region and may be circumstantial evidence for spontaneous osteogenic stem cell differentiation (Caplan AI 2007, Shive MS et al. 2014). Persistent bone marrow edema as a manifestation of the mechanical insufficiency of the injured subchondral bone, in combination with the biomechanical restrictions of fibrocartilage, are the main reasons for the histologically and clinically evident problems of this method (Gomoll AH, Minas T.2014, Steinwachs MR et al 2008). Multiple studies show the limited successful outcomes of MFx for patients >40 with large >3-4cm² defects. Seeking clarity regarding the efficacy of MFx, a recent Cochrane review of Gracitelli GC et al. 2016 examined the evidence from randomized trials comparing MFx to other treatment options. The conclusion was that the currently available evidence is not enough to determine whether mosaicplasty, allograft transplantation, or MFx is better for treating cartilage defects in adults. The long-term results of MFx established a failure rate of about 25% at 10 years requiring an additional intervention.

The Autologous Matrix Induced Chondrogenesis (AMIC®) technique involves the debridement of the cartilage lesion, the classic MFx procedure with ale and the coverage of the lesion site by the collagen membrane Chondro-Gide® plus fibrin. This technique has been used to complete the MFx-cartilage repair process in several joints (knee, hip, talus and metatarsal) so far. Although two recent studies on the use of AMIC® for the repair of articular cartilage defects have been published recently (Gao L et al.2017, Shaikh N et al. 2017). These papers do not exclusively address the knee joint, but rather refer to all three major joints where AMIC® is used (hip, knee, talus). In our meta-analysis (Steinwachs M et al ICRS 2019) the AMIC® procedure significantly improved the clinical status and functional scoring versus preoperative values. Evidence was obtained in a non-selected patient population, corresponding to real-life treatment of knee chondral and osteochondral defects

Content

The idea for a modification of AMIC technology is based on new biological findings related to the tissue healing cascade. Experimental data of the utilization of K-Wire instead of ale for bone marrow stimulation was able to claim superiority in comparison to the conventional microfracture technique (Chen H et al. 2011). Experimental studies with PRGF-Endoret® demonstrate significant stimulation of angiogenesis, stem cell migration, cell proliferation, reduced inflammation and enhanced paracrine cell activity combined with antimicrobial activities in different tissues (Anitua E et al. 2007, Andia I et al. 2012). In addition, PRGF can avoid the activation of the NF-κB pathway, thus reducing the inflammatory and catabolic responses (Jia J et al. 2018). Clinical and experimental data show that the additional use of PRGF (Endoret®) enhances the biological tissue regeneration in bone and cartilage naturally (Filderado G et al. 2011, Anitua E et al 2013). Nowadays, the treatment scarcely addresses subchondral bone as one of the most relevant factors for the outcome of cartilage repair. Based on these new findings we have developed a new AMIC® technique with an enhanced approach to bone marrow stimulation employing tapered 1.5 mm K-Wires with a perforation depth of 1 - 1.5 cm and the subchondral injection of activated autologous PRGF® (Endoret®) in combination with a PRGF derived fibrin clot loaded ChondroGide® collagen membrane (Biological-Enhanced-AMIC;“BE-AMIC”) as a treatment option for symptomatic full-thickness cartilage lesions (> 2 cm²) with bone marrow edema. Initial clinical and MRI data show encouraging results.

Introduction

Focal symptomatic articular cartilage defects greater than 1.5 cm in diameter, if left untreated, may progress to osteoarthritis, which represents the progressive decline of the articular surfaces of the weightbearing joints. Cartilage injuries are common, affecting around 60% of people. Their incidence is increasing and this emphasises the need for the early detection of cartilage defects and the development of new repair and regeneration strategies. No surgical technique has shown conclusive superiority among others. Chondrocytes have a limited potential for replication or migration to a site of injury. Therefore, since joint preservation and regeneration are a worldwide major goal for orthopaedic surgeons, new techniques are being proposed. These new procedures are based upon autologous tissues and mesenchymal stem cells withdrawn from bone marrow or adipose tissue, with the goal that these cells will be able to diffentiate and replicate the anatomic architecture of the osteochondral unit and inherently withstand or otherwise restore resiliency of the cartilage to biomechanical forces, while promoting anti-nflammatory effects across the articular environment, through their paracrine action, thus, finally, preventing morbidity and maintaining mobility of the aging population. Adipose tissue serves as a reservoir for MSCs. Adipose-derived MSCs can be recovered from liposuction aspirates, but their collection from human infrapatellar fat pads has also been reported. These cells have been proved to have the the potential for differentiation into cartilage, as well as bone, tendon, and muscle. The incorporation of autologous ADMSCs into scaffolds with surgical implantation has shown to be successful in the repair of full-thickness chondral defects, while the direct injection of ADMSCs has demonstrated improvement in clinical pain and function outcomes without major adverse events. Goal of our study was to assess the efficacy of the LIPO-AMIC technique (autologous matrix-induced chondrogenesis + ADSCs and adipose tissue transfer) consisting of osteochondral lesion accurate debridement, microfracturing and filling of the defect with bilayer cell-free collagen scaffold soaked in adipose regenerative product.

Content

Eighteen patients (age range: 28-58) were initially treated for symptomatic full thickness knee chondral defects using the LIPO-AMIC technique and followed prospectively for five years. After diagnostic knee arthroscopy, the first step is, under local anesthesia, the extraction, using the simple liposuction method, of 50-80 ml of lipoaspirate from the adipose tissue of the abdomen, using a dedicated single-use kit available on the market for suction and subsequent processing (filtration and micro-fragmentation) and adipose tissue grafting (Process Kit Lipogems, Lipogems International SpA, Milan, Italy), for which use we have followed the instructions provided by the manufacturer. This device progressively reduces the size of the adipose tissue clusters, while at the same time eliminating the blood residues and the oily substances, with pro-inflammatory capacity, minimizing, at the same time, thanks to the completion of the whole process just within saline solution, the risks of damaging the mesenchymal cells. The fragmentation of adipose tissue consists in different steps that make it possible to obtain purified adipose clusters product ready for the desired clinical purpose.

The second step in the surgical procedure is the repair of the focal chondral or osteochondral defect. Once identified, the chondral or osteochondral defect is carefully cleaned up to obtain stable, clean and perpendicular margins of healthy cartilage and then accurately measured. Then, a perfect copy of the defect is obtained using an aluminium model, from which the exact imprint of the defect is cut out. After carefully performing the microfractures of the subchondral bed of the defect, and having exactly cut the membrane (Chondro-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland) on the basis of the model, the collagen matrix, which was immersed for a few minutes in the microframmented adipose tissue and in the stromal vascular fraction extracted from the adipose tissue of the abdomen, is inserted in articulation to directly cover the defect. The remaining part of the cells obtained from the adipose tissue is injected onto the lesion site and then sealed with fibrin glue (Tissucol, Baxter, Rome, Italy). All patients were clinically evaluated through IKDC, KOOS and VAS scores, with follow-up between 12 and 60 months. MRI examinations were performed at 6, 12 months and yearly thereafter. ADSCs have been isolated and characterized in terms of viability and cell composition using multicolor FACS analysis.

RESULTS

No complications during or after surgery were encountered. Patients showed relevant, immediate and durature improvement of the various scores already from the very initial follow-up. At intermediate and final follow-up all scores were significantly increased (p<0.001). MRI examination, completed by T2 mapping imaging, showed early subchondral lamina regrowth and progressive maturation of the repair tissue. Histological studies shown that stem cell population resided in a perivascular location (niche) with preserved architecture and where ADSCs coexisted with pericytes and endothelial cells. FACS analysis confirmed high viability and an increased percentage of endothelial cells.

DISCUSSION

Kramer et al., in 2006, demonstrated in an in vitro study that a collagen membrane is able to treat mesenchymal cells deriving from bone marrow after microfractures and that these cells have the ability to differentiate towards chondrogenic, adipogenic and osteogenic line. Dickhut et al. have performed in vitro studies demonstrating that a biphasic carrier of type I and III collagen, such as the Chondro-Gide® membrane, is able to promote the chondrogenesis of mesenchymal cells. Compared to a collagen-free membrane, it provides greater stability of the repairing tissue.

Mesenchymal stem cells (MSCs) have shown much promise with respect to their use in cartilage tissue engineering. MSCs can be obtained from many different tissue sources. Among these, adipose tissue-derived stem cells (ADSCs) have recently been under investigation as a source for cell therapies. Adipose tissue is very rich in capillary beds, thereby harboring one of the largest depots of MSCs. Adipose tissue can, in fact, provide an abundant source of ADSC’s and stromal vascular fraction and adipose tissue cells. ADSC’s have the advantage of being abundant, easy of harvest, with rapid expansion, high proliferation potential and their ability to better maintain their phenotype with respect to BMSCs. Preparation of ADSCs is also easier and less expensive than BMSCs, and the cultured lineage has shown a similar function to BMSCs in multi-lineage differentiation. For our purposes, the main interest in ADSC’s comes from the fact that these cells are multipotent, therefore able to differentiate into different types of cells including chondrocytes. The chondrogenic potential of human ADSCs has been clearly demonstrated over several years by different works. In 2002, following an in vitro differentiation process, Erickson et al. have shown that human ADSCs maintain the phenotype of chondrocytes and form cartilaginous tissue when implanted subcutaneously in vivo in immuno-deficient mice for up to 12 weeks. In numerous other studies ​​it has been shown that, under certain conditions, ADSCs can express the genes and proteins of different molecules that are specific to cartilage, including type II collagen and aggrecan, without expression of hypertrophic chondrocytes markers, such as type X collagen. However, under various defined conditions, ADSC’s may be induced to synthesize type I and type II collagen, suggesting that a cartilaginous phenotype is also possible. When kept in pellet culture or encapsulated in alginate beads and cultured with 10 ng / ml of TGF-β1 growth factor, ascorbate and dexamethasone, and have been shown to express a chondrocyte-like phenotype and synthesize type II collagen, aggrecan, link proteins and chondroitin sulfate in a time-dependent manner based on the analysis, immunoistochemistry, and the incorporation of mRNA radiotracers, with a significant increase in the synthesis of proteoglycans and proteins under condogenic conditions. Winter et al. concluded that the gene expression profile of ADSCs in chondrogenic conditions is similar to that of mesenchymal cells. Mesenchymal cells from adipose tissue at this time can, therefore, be considered to be able to form cartilaginous tissue.

The Chondro-Gide® is a resorbable collagen membrane, consisting of collagen types I and III. It’s particularity is due to it’s bilayer structure, with one compact and one porous side. The compact layer, which is cell occlusive, prevents cells from diffusion and also protects them from mechanical impact. The porous layer is made of collagen fibers in a loose, porous arrangement that favours cell invasion and attachment. This 3D collagen matrix stimulates the autologous cultured cells to differentiate into the chondrocyte phenotype and to produce collagen II and GAG. The advantage of adding adipose tissue transfer containing ADSC’s is the capability to activate the chondrocytes with regard to outgrowth and proliferation. Vice versa, the cartilage cells might favour the stem cells to differentiate along the chondrogenic lineage.

CONCLUSION

Repair of full-thickness cartilage injuries by LIPO-AMIC technique provides good to excellent clinical improvement, MRI defect filling and, at advanced and detailed histologic evaluation, high percentage of ADSCs and endothelial cell populations with high viability and niche preservation. Results resulted improved in respect to standard AMIC technique and comparable to MACI, but at significantly reduced costs. In conclusion, the LIPO-AMIC technique is an effective and safe single-step cell-based procedure to manage full-thickness focal chondral lesions of the knee.

References

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Introduction

Meny studies have demonstrated the importance of the presence of the meniscus for the knee functioning and have also proven that surgeons should preserve as much meniscal tissue as possible. Not only complete, but also partial, meniscectomy is associated with early degenerative osteoarthritis. To preserve the function of the knee joint, it is now being suggested that meniscal tears should be treated by meniscal repair instead of meniscectomy whenever possible. Among the methods currently used to repair the meniscus, the ideal solution has not yet been found.

Biological augmentation techniques and meniscus tissue engineering strategies are being devised to enhance the likelihood and rate of healing in meniscus repair. Preclinical and clinical studies have shown that the introduction of cellular elements of blood, bone marrow, and related growth factors have the potential to enhance meniscus repair.

Tissue adhesives hold great potential to replace, or support, sutures and staples. Many new adhesive materials, with a good prognosis for use in a variety of applications, have been developed. However, most of them have not been sufficiently characterized to be able to be qualified for meniscus repair

Content

The purpose of this presentation is to present a 5 year follow up of clinical and MRI results of patients having been treated with a newly developed, fully arthroscopic technique of collagen matrix-based meniscus repair, and the injection of liquid bone-marrow into the area of the meniscal lesion. The second aim of this study was to estimate the cost effectiveness of collagen matrix-based meniscus repair arthroscopic technique (AMMR) in Poland in the both Polish National Health Service (PNHS) and in perspecteve of private patients (PP).

54 consecutive patients operated on from 2010 to 2011 with the mean follow up 5,92- year (3,56-8,34) were included. We lost to follow up 6 patients so to final analysis was included 48 cases. Functional scores (IKDC, Lysholm) were assessed preoperatively, 2-year, and at the last follow-up. EQ 5D 5Lscore were assessed at the last follow-up. The state of the meniscus and the total WORMS OA score was also evaluated in MRI.

48 patients were included to the final analysis. Four meniscus were partly resected to the last follow up - 8,3% failure rate. The functionality of the knees improved from preoperative to 2 –year follow up and were even better at 5-year follow up in all the scores used (P < .001). Thirty-seven patients were examined by MRI 2 and 5 years postoperatively. MRI revealed a non-homogeneous signal without meniscal tear in 2-year follow up in 92% and in 5-year follow up in 81% of the operated menisci. The total WORMS score increased from main 6,89 in 2-year follow up to main11,1. No progression to the OA level was observed. Sex, age and BMI have no correlation with 2- and 5-year outcomes. The time from injury to the surgery correlated with clinical. The EQ-5D-5L was0,943.

It is estimated that AMMR decreases the probability of a lifetime replacement of the knee joint by 50% compared to ME and increases QALY by 0.21 (4.71 vs. 4.5). The incremental cost of 8869PLN (perspective of PNHS) and 5619PLN (perspective of PP) for AMMR reflecting reduced resource use balancing part of the costs of the procedure. These values ​​gave a cost of QALY obtained in the amount of 1862PLN (perspective of PNHS) and 1180PLN (perspective of PP).

The 2- and 5-year follow-up data demonstrate that this technique is safe with acceptable failure rate and can offer an additional tool to save the meniscus in the patients otherwise scheduled for meniscal removal. The quality of life 5 year after surgery is very good. The procedure protects the knee against the progression of degenerative.

AMMR is estimated to provide substantial patient benefits over a 5-year horizon, with a considerable increase in QALYs. Despite the increase in costs, the procedure is cost effective at standard thresholds used in the Poland for both analised perspective.