Soluble Collagen VI treatment enhances mesenchymal stem cells expansion for engineering cartilage

Abstract Bone Marrow‐derived mesenchymal stem cells (BM‐MSC) are an attractive source for cell‐based therapies in cartilage injury owing to their efficient differentiation into chondrocytes and their immune‐suppressive abilities. However, their clinical use is hampered by a scarcity of cells leading to compromised efficacy. While expansion of human MSC ex vivo can potentially overcome the scarcity of cells, current methods lead to a rapid loss of the stem cell properties. In this study, we report soluble Collagen VI (cartilage pericellular matrix component) as a potential biologic that can expand the MSC population while maintaining the stem cell phenotype as confirmed by expression of the stem cell markers CD105 and CD90. Short‐term treatment with Collagen VI additionally retains the potential of MSC to differentiate into mature chondrocytes in pellet culture. Cartilage pellets generated from MSC treated with Collagen VI or control express comparable amounts of the chondrogenic markers Collagen II, Aggrecan and Sox9, and the extracellular glycosaminoglycans. Our observations confirm that the use of the endogenous and cartilage‐specific factor Collagen VI is valuable for a rapid and efficient expansion of MSC for potential use in cartilage regeneration and osteoarthritis.


| I N TR ODU C TI ON
Osteoarthritis (OA) is a multifactorial disease that affects the articular cartilage causing deterioration of the joint function. Cartilage injuries and trauma are difficult to repair even in young adults due to the poor regenerative potential of the cartilage tissue, and greatly accelerates OA development. 1 Current treatments of OA are mainly palliative and aim at pain and symptoms management rather than disease modification. 2 Lack of any disease modifying OA drugs (DMOADs) calls for new and effective therapies to repair and regenerate damaged articular cartilage, and to restore joint homeostasis to delay OA progression.
Current cell-based clinical therapies for cartilage repair include the use of autologous chondrocytes or autologous cells from mesenchymal tissues. Stimulation of the endogenous stem cell populations to repair cartilage injuries through microfracture leads to inefficient regeneration and formation of fibrocartilage tissue rather than hyaline cartilage. 3 Autologous articular chondrocyte implantation (ACI) has been clinically approved but the applications have been limited by a paucity of cells and production of functionally inferior fibrocartilage. Mesenchymal stem cells (MSCs) and adipose-derived stem cells (ADSCs) have been investigated to engineer and repair cartilage in basic and early translational studies. MSCs are used for treatment of multiple diseases for their ability to differentiate toward multiple cell types including chondrocytes, bone, and adipocytes, combined to their capacity to secrete anti-inflammatory factors that mitigate inflammation. 4 The role of inflammation in the development of OA is being increasingly recognized. 5 As such, the beneficial anti-inflammatory effects of MSC in prevention or delay of the onset of OA is of substantial interest. Currently, MSCs are being explored as a treatment modality for multiple disease states in hundreds of Phase I and II clinical trials. 6  overcome this limitation and different signaling pathways like the FGF or Wnt can be modulated to maintain the stem cell characteristics of MSCs. [7][8][9] Since these signaling pathways can have multiple effects on tissues other than cartilage, there is a high clinical relevance for the identification and development of biologics that are intrinsic to the cartilage tissue and hence can be more specific.
It is widely accepted that both the extracellular matrix (ECM) and the pericellular matrix (PCM) play a critical role in cartilage function especially for maintaining its biochemical and biomechanical properties. 10 Our recent studies have demonstrated ECM proteins to be a major differential between juvenile and adult cartilage suggesting that the ECM interactions with the chondrocytes also regulate the regenerative capacity of cartilage. 11 Therefore, the emerging understanding of the crosstalk between chondrocytes and ECM or PCM components is required and should be taken in consideration for cartilage tissue engineering. Collagen VI (Col VI) is a major constituent of the chondrocyte PCM consisting of three major a-chains, a1, a2, and a3, along with alternate subunits a4, a5, or a6 that can substitute for a3. 12,13 We have previously demonstrated that short-term treatment of human chondrocytes with soluble Collagen VI (Col VI) and not Collagen I, can increase their number without adversely affecting their ability to generate cartilage. 14 As an extension of that study, we aimed to use Col VI for the expansion of the MSC population preserving their stem cell properties and maintaining their potential to efficiently differentiate into cartilage. and pellets were maintained in culture for 21 days. 0.5 ml of freshly prepared complete chondrogenic medium was replaced every 2 days by complete aspiration of the medium carefully avoiding the pellet.

| Collagen VI treatment and cell growth
Collagen VI (BD 354261) (BD Biosciences, San Jose, CA) was dissolved in a 1.25 mM Tris solution. hMSCs were plated in monolayers at 2,000 cells per well in duplicates in 96-well plates and cultured for 24 hr in complete medium. After 24 hr, cells were treated with control Tris or medium containing 2.5 lg/ml of recombinant human Collagen VI, with media and recombinant protein replacement every day for 4 days. Cell viability was assayed daily with the PrestoBlue Cell Viability Reagent kit (Life Technologies, Carlsbad, CA) as previously described. 14 Fluorescence intensity of the PrestoBlue reagent reduced by living cells was measured at 690nm (650nm excitation wavelength) with a microplate reader (Molecular Devices, Sunnyvale, CA).

| Gene expression analyses
For the monolayer cultures, total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) and for pellet cultures, total RNA was obtained using TRIzol (Invitrogen, Carlsbad, CA). RNA from each sample was reversed transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and quantitative PCR was performed using TaqMan gene-specific expression arrays for Type II collagen-chain alpha 1 (Col2a1) (Hs00264051_m1), Sox9

| Statistical analyses
Data are reported as mean 6 standard deviation (SD). Statistical significance of data was determined by applying a two-tailed Student's t test and p values less than .05 are reported as significant.

| Collagen VI (Col VI) treatment enhances hMSC proliferation
In the present study, we aimed to study the direct effect of soluble Col VI on human bone marrow-derived MSCs (hMSC) fate and function. A quantitative fluorescence-based assay reflecting the metabolic activity of live cells (Prestoblue, Life technologies) was used to measure cell growth, as described previously. 14 In this assay, the reagent resazurin is nonfluorescent but is converted to fluorescent resorufin in the reducing environment of living cells, allowing a quantitative measurement of relative cell numbers. The advantage of this reagent is both high sensitivity allowing detection of relatively low cell numbers reproducibly as well as minimal toxicity such that the reagent can be used at regular intervals during continuous cell culture. As shown in Figure 1

| Col VI treatment retains the chondrogenic differentiation potential of hMSC
To assess if the Col VI treatment retains the potential of hMSC to differentiate into mature chondrocytes and engineer cartilage, we

| D I SCUSSION
Although the benefits of MSC in cartilage tissue engineering have been investigated for many years, more recent studies highlight the benefits deposit an equivalent amount of total GAGs than the untreated control cells. Therefore, the Col VI-expanded MSC can effectively engineer cartilage tissue, comparable to MSC that have not been treated with Col VI but with the advantage of being higher in number. Although the precise mechanism through which soluble Col VI enhances MSC proliferation is not clear, it has been shown previously that the skeletal muscle stem cells self-renewal is profoundly reduced in Col6 2/2 mutant mice. 22 Engraftment of wild-type Col6 1/1 fibroblasts in the mutant mice muscles was able to rescue the defect in stem cell self-renewal leading the authors to conclude that a modulation of the biomechanical properties of the muscle tissue influenced the stem cell self-renewal. In a similar fashion, absence of Col VI in Col6 2/2 mice affects the biomechanical  21 We therefore speculate that Col VI may modulate the cell stem proliferation through interacting with surface receptors that likely sense the biomechanical properties of the microenvironment. The identity of these receptors and the downstream signaling pathways will be further investigated in future studies.
In conclusion, in this study we have described a useful and effective method for MSC expansion utilizing a cartilage-specific factor, Col VI.
We envision that Col VI and similar biologics are promising candidates to enhance the effectiveness of MSC-based therapeutic approaches.

CONFLICT OF INTEREST
The authors declare no competing interests.