Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs

Abstract

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs.

Figure 1

Characterization of ovine MSCs and proliferation and BMP2 transduction of ovine and porcine MSCs: ovine MSCs (a) osteogenic differentiation is seen through dark staining of calcium depositions by Von Kossa silver nitrate staining. (b) Chondrogenic differentiation with chondroitin sulfate proteoglycans stained blue with Alcian Blue staining, and (c) adipogenic differentiation as seen through intracellular lipid staining with Oil Red O. (d) Ovine A, ovine B, and porcine cell line proliferation rates. (e) Proliferation rates of ovine A and ovine B following transduction with 15,000 vp/cell of adenoviral BMP2. (f) Transduction with 15,000 vp/cell adenoviral BMP2 production from ovine and porcine MSCs both with and without cryopreservation.

Figure 2

Viability of MSCs on day 0 and 4 after encapsulation with and without cryopreservation: MSCs were stained with calcein AM for live (green) and ethidium homodimer for dead (red). On the day of microencapsulation ovine A MSCs stained for (a) live, (b) dead, and (c) overlay. And on day 4 following microencapsulation (e) ovine A MSCs stained for live, (f) dead, and (g) overlay. (d) Graphical representation of counts of 90 images. Cryopreserved ovine A encapsulated MSCs on day of thaw (i) live, (j) dead, and (k) overlay. Day 4 postthaw ovine A MSCs stained for (l) live, (m) dead, and (n) overlay. (h) Graphical representation of counts of 90 images (averaging 87,000 cells per group). (*P < 0.0001).

Figure 3

BMP2 production in microencapsulated MSCs. (a) Cells were transduced with 15,000 vp/cell adenoviral BMP2 prior to microencapsulation and plated out freshly or cryopreserved. (^{+, #} P < 0.01), (b) ovine A and ovine B cryopreservation and BMP2 transduction effect on MSC viability on day of microencapsulation (day of thaw for cryopreserved samples), and (c) 4 days after microencapsulation (P < 0.05).

Figure 4

Structural analysis of freshly prepared and cryopreserved encapsulated MSCs: (a) phase contrast of microencapsulated ovine A MSCs showed clear borders on microbeads. (b) SEM of MSC microbeads showed a uniform surface. (c) SEM of MSC microbeads of all sizes showed uniform structure. (d) Phase contrast of cryopreserved microencapsulated MSCs did not show appreciable damage. (e) SEM of cryopreserved MSC microbeads showed a uniform surface. (f) SEM of cryopreserved microencapsulated MSCs showed no damage to beads of various sizes.

Figure 5

BMP2-transduced microencapsulated MSCs bone formation in a mouse model for heterotopic ossification. 3 × 10⁶ ovine B MSCs transduced with 15,000 vp/cell were injected into the hind limb of a NOD/SCID mouse. The resulting heterotopic ossification was observed by X-ray and MicroCT for (a) and (b) for freshly prepared BMP2 microencapsulated MSCs and (d) and (e) for cryopreserved BMP2 microencapsulated MSCs. (c) The volume of the resulting heterotopic ossification was not different between the two groups.