Coupling Osteogenesis and Vasculogenesis in Engineered Orthopedic Tissues

Abstract

Inadequate vascularization of engineered tissue constructs is a main challenge in developing a clinically impactful therapy for large, complex, and recalcitrant bone defects. It is well established that bone and blood vessels form concomitantly during development, as well as during repair after injury. Endothelial cells (ECs) and mesenchymal stromal cells (MSCs) are known to be key players in orthopedic tissue regeneration and vascularization, and these cell types have been used widely in tissue engineering strategies to create vascularized bone. Coculture studies have demonstrated that there is crosstalk between ECs and MSCs that can lead to synergistic effects on tissue regeneration. At the same time, the complexity in fabricating, culturing, and characterizing engineered tissue constructs containing multiple cell types presents a challenge in creating multifunctional tissues. In particular, the timing, spatial distribution, and cell phenotypes that are most conducive to promoting concurrent bone and vessel formation are not well understood. This review describes the processes of bone and vascular development, and how these have been harnessed in tissue engineering strategies to create vascularized bone. There is an emphasis on interactions between ECs and MSCs, and the culture systems that can be used to understand and control these interactions within a single engineered construct. Developmental engineering strategies to mimic endochondral ossification are discussed as a means of generating vascularized orthopedic tissues. The field of tissue engineering has made impressive progress in creating tissue replacements. However, the development of larger, more complex, and multifunctional engineered orthopedic tissues will require a better understanding of how osteogenesis and vasculogenesis are coupled in tissue regeneration. Impact statement Vascularization of large engineered tissue volumes remains a challenge in developing new and more biologically functional bone grafts. A better understanding of how blood vessels develop during bone formation and regeneration is needed. This knowledge can then be applied to develop new strategies for promoting both osteogenesis and vasculogenesis during the creation of engineered orthopedic tissues. This article summarizes the processes of bone and blood vessel development, with a focus on how endothelial cells and mesenchymal stromal cells interact to form vascularized bone both during development and growth, as well as tissue healing. It is meant as a resource for tissue engineers who are interested in creating vascularized tissue, and in particular to those developing cell-based therapies for large, complex, and recalcitrant bone defects.

Keywords: angiogenesis; bone tissue engineering; coculture models; endothelial cells; mesenchymal stromal cells; vascularization.

FIG. 1.

General anatomy of bone. A macroscopic-to-microscopic schematic shows how blood vessels are integrated into bone tissue to support the resident cells in both cancellous and cortical bone. ECs secrete osteogenic factors and osteoblasts secrete angiogenic factors during native bone regeneration. Figure adapted from Lopes et al. and Grosso et al. ECs, endothelial cells. Color images are available online.

FIG. 2.

Pathways of bone formation during development. (A) Intramembranous ossification involves direct differentiation of mesenchymal progenitor cells into an osteogenic lineage. (B) Endochondral ossification involves formation of a cartilaginous template that undergoes hypertrophy, followed by transformation into bone. Figure adapted from Einhorn and Gerstenfeld and Almubarak et al. Color images are available online.

FIG. 3.

Schematic of the regenerative niche following injury. The approximate timing and main stages of fracture healing. The process begins with an initial inflammatory phase and formation of a hematoma at the defect site. Surrounding vasculature invades the fibrous matrix and supplies cells responsible for subsequent callus formation. This cartilaginous callus becomes mineralized and eventually is remodeled, giving rise to new trabecular and cortical bone. Color images are available online.

FIG. 4.

Cellular interactions between ECs and MSCs in blood vessel formation. ECs form the inner lumen of the vessel, while MSCs differentiate into pericyte-like cells and promote maturation and stabilization of vasculature. Figure adapted and used by permission from Melchiorri et al. MSCs, mesenchymal stromal cells. Color images are available online.

FIG. 5.

Osteogenic predifferentiation of MSCs improves bone-forming potential in vivo. BMSCs were expanded in either basal or osteoinductive medium for 3 weeks before seeding on beta-tricalcium phosphate scaffolds and implantation subcutaneously in a heterotypic nude mouse model for up to 20 weeks. Freshly isolated BMNCs were used as a control. Constructs seeded with osteo-induced BMSCs produced more robust bone regeneration, relative to both noninduced BMSC- and BMNC-seeded constructs, as shown by greater implant density quantified from microcomputed tomography images. Scale bar = 5 mm. *p < 0.05 compared with both the noninduced BMSCs and BMNCs. Figure adapted and used by permission from Ye et al. BMSC, bone marrow-derived MSC; BMNC, bone marrow mononucleated cell. Color images are available online.

FIG. 6.

Direct coculture of MSCs with ECs promotes smooth muscle cell differentiation of MSCs. HMSCs cocultured with HUVECs exhibit increased (A) expression of smooth muscle cell differentiation markers and (B) collagen genes. Removal of MSCs from coculture resulted in decreased expression of these genes over time, and (C) an increase in mesenchymal stemness markers. *p < 0.05 relative to control without cocultured ECs. Figure adapted and used by permission from Lin and Lilly. HMSC, human bone marrow-derived MSC; HUVEC, human umbilical vein endothelial cell.

FIG. 7.

Effect of culture medium on vessel development and osteogenic differentiation. EC elongation over 2 weeks in culture is clearly affected by the nutrient medium: (A) osteogenic medium, (B) vasculogenic medium, (C) combination of osteogenic and vasculogenic media. CD31 staining (green) demonstrated the presence of vessel-like structures and DAPI staining (blue) identifies all cell nuclei present in cultures. Similarly, culture medium affects the expression of osteogenic markers by MSC over 2 weeks in culture: (D) Alizarin Red, (E) ALP activity. Scale bar = 150 μm. *p < 0.05 compared with other groups. Figure adapted and used by permission from Kolbe et al. DAPI, 4′,6-diamidino-2-phenylindole; ALP, alkaline phosphatase; ODM, osteogenic differentiation medium; EGM2, vasculogenic medium; ODM-S_EC, osteogenic medium with endothelial supplements; EGM2-βGly, vasculogenic medium supplemented with β-glycerophosphate; EGM2-S_ODM, vasculogenic medium supplemented with β-glycerophosphate, ascorbic acid, and dexamethasone. Color images are available online.

FIG. 8.

The process of endochondral ossification. Initial MSC differentiation into chondrocytes and condensation is followed by a hypertrophic phase with vascular invasion. The intermediate cartilaginous template undergoes remodeling and mineralization, and further remodeling to produce mature bone. Figure adapted and used by permission from Freeman and McNamara. Color images are available online.

FIG. 9.

Chondrogenic and osteogenic priming of engineered bone constructs. MSC seeded on collagen/hydroxyapatite scaffolds were primed for 5 weeks in osteogenic or chondrogenic medium before implantation in a rat critical-sized calvarial bone defect model. Chondrogenically primed constructs showed more mature bone formation [(A–C), areas of newly formed bone are highlighted by blue arrows], and enhanced blood vessel formation (D). *p < 0.05 statistically significant difference between control and other groups at 8 weeks, **p < 0.05 statistically significant difference between 4 and 8 weeks of chondrogenically primed group. Figure adapted and used by permission from Thompson et al. Color images are available online.

FIG. 10.

Chondrogenic priming, media, and coculture of MSCs and ECs influence osteogenic and vasculogenic potential of engineered constructs. MSC pellets were primed in chondrogenic medium (CP) for 3 weeks and were then continued as monocultures (−HUVECs), or were seeded with ECs (+HUVECs) or both ECs and MSCs (+HUVECs:MSCs) before being cultured in vasculogenic medium alone (−OM) or in combination with osteogenic supplements (+OM) for an additional 3 weeks. (A) CD31 staining (green) demonstrated the presence of integrated blood vessels in the group seeded with both ECs and MSCs, but not in the other groups. All cell nuclei were counterstained with either DAPI (blue) or Propidium Iodide (red). White boxes denote examples of the rudimentary vessels present within the aggregates. Quantification of osteogenic markers reveals enhanced (B) ALP activity and (C) calcium content in chondrogenic cultures that were seeded with HUVECs and MSCs. ^p < 0.05 versus CP21 + HUVECs group, ^ap < 0.05 versus CP21 − HUVECs, ^bp < 0.05 versus Osteo alone. Figure adapted and used with permission from Freeman et al. CP, chondrogenically primed; −OM, no osteogenic supplements. Color images are available online.