A Modular Approach to Generating Tissue Engineered Bone
Kevin B. Miles1, Tristan Maerz2, and Howard W. T. Matthew1
1
2
Chemical Engineering & Materials Science Department, Wayne State University, Detroit, MI.
Orthopaedic Research Laboratories, Beaumont Hospital, Royal Oak, MI.
Modular tissue engineering involves the construction of large (macro-scale) tissue structures from
smaller (micro-scale) tissue units or modules. The field has the potential to solve various cell-density
related challenges associated with traditional tissue engineering. Previous studies have demonstrated the
value of vascularization enhancing strategies as a necessary component in bone defect regeneration. In
this study, we investigated the fabrication of macro-scale, bone-like constructs by fusion of micro-scale
modular units composed of natural polyelectrolyte polymers, hydroxyapatite (HAP) granules, and
mesenchymal stem cells (MSCs). The micro-scale modules were formed by complex coacervation
whereby an ionic reaction between chondroitin 4-sulfate (C4S) and chitosan produced an insoluble
polyelectrolyte membrane. Briefly, a C4S solution with suspended MSCs and HAP granules was
extruded as 400 micron diameter droplets and collected into stirred chitosan solution. The insoluble
polyelectrolyte complex was formed at the droplet-chitosan interface, encapsulating MSCs and HAP
granules suspended in the C4S solution. The resulting microcapsule modules were transferred to dish
culture, and maintained in a standard osteogenic culture medium. Analysis of the cultured modules over
time indicated that C4S/chitosan/HAP microcapsules supported MSC proliferation and differentiation to
an osteoblastic lineage. Osteoblastic activity was assessed by quantification of: bone-specific protein
secretion (osteocalcin, osteopontin, collagen), calcified matrix deposition, and increases in alkaline
phosphatase (ALP) activity. Moreover, the micro-scale modules could be fused post-culture to create
larger (1.5 cm3) tissue constructs which possessed inherent pore spaces between the spherical,
mineralized units. Fusion was accomplished by generation of additional polyelectrolyte complex
between packed modules using a sequential polymer perfusion procedure. The compressive strength and
elastic modulus of fused tissue constructs increased significantly during induced MSC osteogenesis,
compared to values for constructs contain non-differentiating MSCs: fused constructs fabricated from
microcapsules containing MSCs undergoing osteoinduction for 4 weeks exhibited a compressive
strength of 6.2 MPa (+ 3.1 MPa), significantly higher than control fused constructs without
differentiating MSCs (0.0059 + 0.0011 MPa). Moreover, microCT data shows that differentiating MSCs
are able to extensively mineralize the walls and interior of C4S/Chitosan microcapsules: microcapsules
containing MSCs osteoinduced for 4 weeks, but no initial HAP granules, exhibited a bone volume
fraction of 182.5 mg HAP/cm3, compared to a bone volume fraction of zero for capsules without
differentiating MSCs or HAP granules included initially in capsule fomration. Inter-module pores were
found to facilitate prevascularization of constructs by culturing accessory cells, such as endothelial cells,
on the exterior of the modules prior to fusion. Current studies are evaluating the effects of alternate
fusion chemistries on construct mechanics and the physiology of encapsulated and accessory cells.
Results to date indicate that the C4S/chitosan/HAP based module system can form the basis of either a
3D printing based or an injectable matrix based strategy for generation of vascularized bone.