Hydrogel Microsphere Encapsulation of a Cell-Based Gene Therapy System Increases Cell Survival of Injected Cells, Transgene Expression, and Bone Volume in a Model of Heterotopic Ossification

Abstract

Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid endochondral bone formation, enhancing our preexisting cell-based gene therapy system by microencapsulating adenovirus-transduced cells in nondegradable poly(ethylene glycol) diacrylate (PEGDA) hydrogels before intramuscular delivery. This study evaluates the in vitro and in vivo viability, gene expression, and bone formation from transgenic fibroblasts encapsulated in PEGDA microspheres. Fluorescent viability and cytotoxicity assays demonstrated >95% viability in microencapsulated cells. ELISA and alkaline phosphatase assays established that BMP-2 secretion and specific activity from microencapsulated AdBMP2-transduced fibroblasts were not statistically different from monolayer. Longitudinal transgene expression studies of AdDsRed-transduced fibroblasts, followed through live animal optical fluorescent imaging, showed that microencapsulated cells expressed longer than unencapsulated cells. When comparable numbers of microencapsulated AdBMP2-transduced cells were intramuscularly injected into mice, microcomputed tomography evaluation demonstrated that the resultant heterotopic bone formation was approximately twice the volume of unencapsulated cells. The data suggest that microencapsulation protects cells and prolongs and spatially distributes transgene expression. Thus, incorporation of PEGDA hydrogels significantly advances current gene therapy bone repair approaches.

Bioencapsulation Technologies in Tissue Engineering

“…PEG and its derivatives, e.g., poly(ethylene glycol) diacrylate [PEGDA], have been widely used in tissue engineering due to their biocompatibility and ability to be altered to physically mimic soft tissues (34, 35). PEG is one of the few synthetic polymers that can be used for both microencapsulation and macroencapsulation (36), and it has been extensively studied for the surface modification of scaffolds, such as vascular grafts, due to its nonimmunogenicity and nonantigenicity (37, 38).…”

Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies

“…Briefly, viral transductions (5000 vp/cell) were done using Dulbecco's modified Eagle's medium containing 2% fetal bovine serum as describe [20]. 5 x 10 6 transduced cells alone or cells encapsulated with poly(ethylene glycol) diacrylate (PEGDA) were used as previously described [12]. Three mice were intramuscularly injected with microencapsulated (N = 2) or unencapsulated (N = 1) transduced cells in the right paraspinous muscle of a mouse lumbar spine.…”

“…Optimal treatment requires stable, high local concentrations of BMP-2 [10][11][12]. Recent case reports suggest that high doses of BMP-2 leads to rapid unwanted events including osteolysis of the bone and is potentially carcinogenic [13].…”

“…Techniques that involve ex vivo transduction of cells with BMP-2 viral vectors circumvent these problems and allow for efficient expression of the BMP-2 at the site specific area. However, the cell based strategy depends on stable cell placement at the site where therapeutic action is needed [12,14]. Recently, we showed that poly(ethylene glycol) diacrylate (PEGDA) hydrogel is an effective carrier for encapsulation of BMP-2 producing cells enabling stable placement and allows for the use of allogenic cells, without launching an immune response that could otherwise limit BMP-2 local release [12].…”

“…However, the cell based strategy depends on stable cell placement at the site where therapeutic action is needed [12,14]. Recently, we showed that poly(ethylene glycol) diacrylate (PEGDA) hydrogel is an effective carrier for encapsulation of BMP-2 producing cells enabling stable placement and allows for the use of allogenic cells, without launching an immune response that could otherwise limit BMP-2 local release [12]. Methods to longitudinally image the placement of PEGDA encapsulated BMP-2 producing cells are needed to predict cell viability and location of optimal ossification and fusion to show preclinical efficacy.…”