Early Regenerative Intervention for Post-Traumatic Osteoarthritis

The field of regenerative medicine and orthobiologics have raised the interests of many clinicians and members of the scientific community. Over the last 20 years there has been a great interest and growth in this field, a Scopus search for musculoskeletal and stem cells resulted in over 3000 documents, with more than a third being reviews. A PubMed search in the past 6 years resulted in 52 publications in the last 5 years.

Unfortunately, the fragmentation of regenerative medicine has occured with many clinicians using generalized protocols resulting in little significant clinical improvement or short-term benefits that often fade over time.

There is still a great deal of research which needs to be done to elucidate how best to utilize the various components of regenerative medicine as well as the pathophysiology of osteoarthritis.

The ERIPTO study seeks to study a proposed protocol utilizing various components of regenerative medicine including platelet rich plasma (PRP), hyaluronic acid (HA), and human bone marrow aspirate concentrate (BMAC).

In trauma and normal wound healing collagen is often the initial activator of platelets with development of platelet monolayers over the exposed collagen surfaces as seen in post-traumatic joints. This is opposed to immediate proteolytic cleavage of platelets by thrombin with subsequent immediate release of stored growth factors. Investigations of gene expression within joints following mechanical injury to articular cartilage have been studied. In a shear versus impaction model investigators found an increase in the expression of collagen type 1 following trauma to the articular surface. This expression of collagen may act as an anchor for percutaneously injected PRP in post-traumatic joints to help facilitate steady secretion of growth factors.

It is known that the average life span of a mesangial cell once delivered into a joint is around 30 days. Only 26% of transplanted cells survive within the joint. This time frame is contingent on providing a proper and hospitable environment for the cell to be injected into. In consideration of the catabolic micro-environment of osteoarthritis it is evident that an arthritic joint, many times, harbors a molecular milieu that is not suitable or beneficial to transplanted mesangial cells and native cells including chondrocytes, type 2 collagen, the extracellular matrix, synovium, as well as subchondral progenitor cells. It can also promote osteogenic differentiation of native cartilage and lead to the progression to end stage joint disease.

Research is also coming to light regarding the inflammatory niche present in the environment at the time mesangial cells are transplanted and can hold a bearing in regards to trophic factors secreted by the mesenchymal stem cells (MSCs) as well as the immunomodulatory effects they possess. Toll like receptor stimulation on MSC polarization plays an important role in the development of a pro-inflammatory or anti-inflammatory MSC. Pro-inflammatory factors present in the milieu such as interferon (IFN) gamma or IFN alpha can influence the differentiation down the pro-inflammatory lineage of the MSC and help promote subsequent immune modulation to promote differentiation of the M1 (pro-inflammatory) macrophage, activation of CD8 T cells, as well as increased expression of pro-inflammatory chemokines such as CCL2, CXCL-9, and CXCL-10. [17] If a low level of iNOS is present that can subsequently lead to T cell activation and proliferation via cytokine-induced chemokines. Although this is largely true, expression of MSC induced IL-6 helped promote the differentiation to M2 (anti-inflammatory) lineage.

Recent research has shed light upon a novel concept within post-traumatic and arthritic joints. This is the effect the inflammatory environment and reactive oxygen species can have on native tissue dysfunction, particularly growth factors such as the TGF-B superfamily along with BMP. In 2019 investigators researched native MSC dysfunction with dysfunctional TGF-B possibly as a result of the catabolic milieu in osteoarthritis. TGF-B-induced protein (TGF-Bi) has been seen as a growth factor expressed in MSCs native to joints as well as bone marrow. It has been found to play two roles in normal chondrogenic differentiation.

In one role it stimulates proliferation and differentiation while in another role it inhibits mineralization and chondrocyte terminal differentiation. It is postulated that TGF-Bi in BM-MSCs helps regulate condensation of BM-MSCs in the early phase of chondrogenesis by bridging action between cells, collagens, and proteoglycans. In their study the investigators came to the conclusion this was achieved by modulation of adhesion molecules ITGB1, ITGB5, and NCAD. In their study the investigators were able to demonstrate the counterintuitive upregulation of TGF-Bi within the bone and articular cartilage of joints afflicted with OA, giving rise to the question of deregulation of native growth factors.

The investigators postulated either this could be the result of TGF-Bi attempting to reverse calcific changes occurring in chondrocytes of severely arthritic regions of the knee and noted an increased concentration of such within osteophytes and areas of extensive damage. The investigators also suggested a dysregulation of TGF-B family of genes within OA and did note the regulatory role TGF-Bi plays on collagen X, a marker of chondrocyte hypertrophy. Evidence is also coming to light of multiple MSC niches which exist within joints that assist in maintaining cartilage integrity and helping heal microdefects within damaged cartilage. The investigators noted among the MSCs, TGF-Bi was down regulated within the infrapatellar fat pad and BM-MSCs from OA joints and noted that whether BM-MSCs efficiency to repair cartilage lesions in vivo warrants further investigation.

The role of reactive oxygen species play in dysregulation of native BMP within musculoskeletal conditions has also been studied. The investigators discussed how low levels of ROS are required to maintain undifferentiated MSCs and how ROS (generated by NOX-2 and NOX-4) and BMP play crucial physiological roles in chondrogenesis. In a discussion regarding osteonecrosis and osteoarthritis the investigators note necrotic lesions lead to the increased production of free oxygen radical (O-, H202) with subsequent increased production of BMP-2, seen in necrotic lesions and damaged cartilage from OA patients. Increased H2O2 levels, produced by either NOX activation or inflammation, induce NF-kB activation which enhances expression of BMP-2. BMP2 stimulates proteoglycan synthesis, induces vascularization, promotes endochondral osteogenesis and has anabolic effects on chondrocyte metabolism and function, and is considered a marker for OA severity. Dysregulation of this system can lead to oxidative stress and cell dysfunction which may contribute to various pathologies.

Also, when considering MSCs capability to promote neocartilage formation, inflammation certainly plays a factor regarding its efficacy. Inflammatory factors such as TNF-alpha, IL-1B, and IL-6 hinder MSCs capability in promoting chondrogenesis by the production of excessive free radicals. The role of IL-1B in contributing to an inflammatory microenvironment has also been evaluated. These include cartilage degradation, induction of Ik-B alpha phosphorylation and subsequent nuclear translocation of nuclear factor - kappa B (NF-kB), increased reactive oxygen species (ROS) production (may also activate NF-kB), increased expression of ADAMTS-4 (inhibits gene expression of aggrecan and COMP) and reduced gene expression of anti-oxidant enzymes. The investigators discussed how elevated oxidative stress produced by these inflammatory cytokines inhibits the synthesis of cartilage matrix macromolecules (i.e. type 2 collagen and aggrecan), promotes the degradation of articular cartilage in arthritic joints, and inhibits native and implanted mesenchymal stem cell chondrogenesis.

It is now known that PRP has various influences on MSCs when combined with MSC treatment including increased proliferation rate of chondrocytes, helping promote chondrogenic differentiation, RUN X 2 inhibition, trapping of NF-KB, and affecting an overall anabolic change in the microenvironment.

It is now known that mesangial cells play an orchestrating role via paracrine related mechanisms in the expression of anabolic factors including expression of type 2 collagen, well documented anti-inflammatory and immunomodulatory role, as well as studies demonstrating recruitment of native progenitor cells versus direct differentiation. This is at the heart of what transplanted MSCs do to help in the healing process. As previously mentioned, there is only a 26% survival rate for transplanted cells and there is significant push to find bio-engineering modalities to help support the survival of transplanted MSCs. Among the modalities include transplantation of MSCs with Hyaluronan derived matrices.

Both CD44 and CD168 are involved in the regulation of growth factor signaling. Implanting MSC within an HA scaffold can help augment communication of MSCs within the joint microenvironment. The investigators also discuss the well documented pleiotropic effects of HA including anti-apoptotic, anti-inflammatory, immunosuppressive, anti-fibrotic, and chondroprotective effects. As mentioned above, with endogenous MSC dysfunction due to the presence of an inflammatory niche within osteoarthritic joints, there is an immunological response which also contributes to the arthritic milieu within joints. HA has demonstrated immunosuppressive qualities and have seen the decrease in the levels of activated T cells. There is also benefit in utilizing commercially available HA in helping normalize endogenous HA synthesis.

With the proposed ERIPTO protocol there is a logical approach to achieve more longer lasting and clinically relevant results.