Nathan Katz, MSc, Ph.D., Jointechlabs, Inc., USA
Avner Yayun, MD, Ph.D., Procore Biomed, Israel
Procore Biomed has developed a hyaluronic-acid – plasma cross-linked hydrogel “RegenoGel” suitable for the growth and differentiation of skeletal tissue stem and progenitor cells.
Jointechlabs has provided adipose-derived cell fraction obtained by the “Mini-Stem System”, which contains a range of 2-8% of mesenchymal stem cells with a potential chondroprotective effect on the joints in patients suffering from Knee OA.
Procore tested several parameters for the growth and differentiation of these cells when embedded in Regenogel OSP-based hydrogels. The hydrogels were prepared using the patient’s own autologous plasma linked to Regenogel-OSP-activated HA. Approximately 1 million cells (corresponding to approximately 40-50,000 MSCs) were mixed with the conjugate in loose-cap 5 ml falcon tubes and embedded within the hydrogel by the addition of Thrombin (approximately 0.25 IU/gel). 2 hrs later growth medium (1 ml; DMEM: F12) was added on top of the hydrogel and the tubes were placed in a 5% CO2 Incubator for different time points.
The integrity of the hydrogel was monitored by visual inspection of the intact hydrogel over time. Survival and proliferation of the cells were monitored using the metabolic dye Alamar blue. Cell differentiation was monitored using a functional ELISA for the presence of VEGF in the culture medium. Chondrogenic differentiation was monitored by measuring soluble glucosaminoglycans (sGAG). The effect of the proliferation and differentiation of these cells within the hydrogel was monitored with or without the addition of 10ng/ml of FGF2 twice weekly as a positive control for MSCs survival and proliferation.
Results:
The differentiation and proliferation of ADCs in Procore’s Regenogel-OSP. A. Proliferation is delayed without FGF2 but reaches similar levels after approximately two weeks. B. VEGF signal begins to rise after approximately one week in culture in the presence of FGF2, and after 12 days in the absence of FGF2, reaching similar levels at 15 days in culture. C. Soluble Glucosaminoglycans (sGAG) increase parallels VEGF and Alamar Blue signal increase.
Conclusions:
Hydrogel integrity: All hydrogels remained fully intact after 15 days in culture, and displayed no signs of degradation.
Cell survival and proliferation: There was excellent survival and proliferation of the cells both with and without FGF2. FGF2 moderately accelerated the proliferation at initial time points (approximately one week in culture).
Secretion of VEGF: VEGF as a major angiogenic stimulator is largely secreted by mesenchymal stem cells (MSCs), and serves as a marker for MSC potency. A sharp increase in VEGF levels produced by the embedded cells indicates good survival and retention of active MSCs within the hydrogel for at least 15 days.
Chondrogenic differentiation: soluble glucosaminoglycans are secreted into the medium by differentiating chondrogenic cells. Indeed, a sharp rise beginning after one week in culture was detected.
We continue to follow up on the integrity, survival, and differentiation of MSCs in the Hydrogel over time.