Particulate and rigid ceramic scaffolds have a role as supports for osteogenic stem cells in bone tissue engineering. Particulate scaffolds, with which our group has greater in vivo
experience, can be combined ex vivo
with culture-expanded BMSCs and placed into animal recipients, where they will successfully form bone within 8 weeks.7,9–13
In nude mice, bone formation can occur with rodent BMSCs or hBMSCs, in either orthotopic or heterotopic locations.7,13
Such transplants have successfully closed critical-sized calvarial defects in mice and dogs.1,7
The loose cohesion between particles appears to facilitate nourishment and eventual vascularization of cells within the entire cross section of these transplants, even those as thick as 8
Despite these attractive features, particulate scaffolds' main drawback is their inability to bear mechanical loads during bone formation, making them poor candidates for immediate load-bearing reconstruction of long-bone defects or fractures.
Rigid ceramic scaffolds, in contrast, can be formulated to bear substantial loads from the time of placement. However, earlier rigid scaffolds have not been successful at promoting bone formation by BMSCs because they were unable to balance the competing demands of porosity and strength. Strong scaffolds, for instance, have been too dense to support cell survival; conversely, porous scaffolds maintain cell viability but cannot support loads. Our group is now attempting to develop novel strong scaffolds that simultaneously support cell survival and bone formation.2,4,14–16
Despite our initial success, actually understanding how to optimize scaffold geometry, structure, and composition remains a formidable challenge, given the myriad number of parameters that can potentially be optimized. This effort has been somewhat simplified by earlier investigators who focused on improving the osteoconductive properties of rigid scaffolds.17
Those studies typically involved placing the scaffold adjacent to normal bone and then measuring the rate and amount of bone ingrowth into the scaffold. Manipulation of HA and TCP ratios, micro-porosity, macro-porosity, and surface texture have optimized the ability of these scaffolds to support new bone ingrowth. Unfortunately, these studies have typically examined bone formation at the surface of the scaffold rather than at its interior, because they occur over short time-frames, so they could not address questions involving penetration of bone into the scaffold interior. Additionally, none of these studies have included co-transplantation of human osteoprogenitor cells, as our study has.18–21
This study was designed to offer several novel sets of observations. First, observations were made over an entire year, rather than the typical 8 to 16 weeks found in most other papers in this field, demonstrating that bone formation continues to increase until at least 38 weeks. Second, histologic and mechanical observations were made synchronously, demonstrating that they are optimized at different pore sizes, and therefore need to both be measured to better describe the effect of pore size on scaffold performance.
This study was also designed to evaluate the interplay between scaffold surface area and the density of hBMSCs within the scaffold on bone formation. New bone formation first occurs on the scaffold surface, so that scaffolds with substantial surface areas would be expected to promote substantial bone. Yet, linear increases in cell density lead to exponential increases in bone formation, perhaps due to paracrine signaling among the cells, so that scaffolds with substantial voids which were filled with cells would be expected to also promote bone formation. Our study attempted to identify which of these conditions (substantial surface area vs. high cell density) might best promote bone formation. The experiments were thus established with an expectation that cell density in the scaffolds would increase as lamellar spacing increased.
In this study, we created via robo-casting a set of HA scaffolds differing only in the distance between adjacent lamellae. The thickness of the lamellae, their texture, and their microporosity were kept consistent from scaffold to scaffold. We cultured hBMSCs onto the scaffolds ex vivo and then transplanted the constructs into nude mice. Scaffolds were observed at time points ranging from 9 to 50 weeks. By histology, bone formation within each scaffold was quantified across the entire cross section and specifically at its interior. In situ hybridization was used to confirm a donor source for the bone and osteoblasts. Scaffold strength was measured in the scaffolds before cell seeding and then at 23, 38, and 50 weeks.
We observed bone formation at the first time-point (9 weeks) at the scaffold periphery. With increasing time, overall bone formation increased steadily, proceeding radially inward. Lamellar spacing of 500
μm was most supportive of bone formation at the latest time-points of 38 and 50 weeks in the overall transplant, and especially supported bone formation within the scaffold interior at the intermediate time-point of 23 weeks. The scaffolds also supported the formation of a marrow organ, consistent with our expectations of a bone-supporting scaffold. The osteocytes within the newly formed bone and the osteoblasts lining the bone were confirmed to be of human origin using in situ
hybridization for Alu
, and were therefore the transplanted hBMSCs or their progeny. No hBMSCs independent of newly formed were identified, and no cartilage was seen in any section. No tumors were observed in the transplants or elsewhere in the animals, consistent with our 14-year experience transplanting hBMSCs into nude mice.
Transplant compressive strength offered additional insights into scaffold performance. Among fresh scaffolds, compressive strength predictably increased as lamellar spacing decreased. Following transplantation and then harvest at the 23-week time-point, bone had developed in all transplants and was associated with increases in compressive strength in all groups. At later time-points, however, the association between greater bone formation and greater compressive strength was lost, in that transplants with narrow lamellar spacing had diminished strength despite an increase bone formation and no change in scaffold volume. The loss of scaffold strength has two possible explanations—it could be due to a weakening of the inherent scaffold structure over time, most likely due to resorption of the more soluble β-TCP from the scaffold, or it might be due to propagation of micro-fractures within the scaffold.22
Distinguishing between these two explanations would require additional testing of the transplants in a fresh experiment, perhaps using X-ray microtomography and SEM to quantify the degree of in vivo
In our current study, the compressive strength of our scaffolds reflected an interplay between bone formation and scaffold resorption. It is perhaps not surprising, then, that scaffold histology and mechanical strength do not necessarily coincide—the transplants with the greatest bone formation (500
μm spacing) were not those with the greatest strength (200
μm spacing). These results highlight the importance of using multiple measures of scaffold success (histologic appearance and mechanical performance) to help predict scaffold performance.
An ideal experimental system would permit us to examine the impact of bone formation on scaffold strength separately from the impact of scaffold resorption. Unfortunately, that is difficult to achieve in our model, in which transplanted BMSCs support both bone formation and the formation of a marrow microenvironment; this new marrow in turn may accelerate the scaffold resorption process by introducing osteoclasts to the scaffold. Thus, bone formation supports hematopoiesis, which in turn supports scaffold resorption. The most obvious method for assessing scaffold resorption in the absence of bone formation might have been inclusion of a cell-free transplant in the mouse experiment. While this transplant would have failed to support new bone formation, it would also have failed to form a marrow microenvironment, so it would not undergo as substantial a degree of resorption as a BMSC-imbued transplant.
Our findings support the hypothesis that lamellar spacing helps determine the amount of new bone that forms on HA scaffolds as well as the compressive strength of these scaffolds after 1 year. We speculate that spacing that is too wide may not provide a sufficiently high density of BMSCs to stimulate bone formation,23
whereas spacing that is too narrow may impede short-term nourishment of the cells and long-term vascular ingrowth. These findings are roughly consistent with those in our prior study using particulate scaffolds, in which particles smaller than 100
μm and larger than 1000
μm promoted markedly less bone formation.10
The investigation of the specific impact of lamellar spacing on vascular invasion, and the impact of vascular invasion on bone formation, might shed light on these speculations. However, we have observed that successful development of bone cannot occur without adequate vascularization. BMSCs do attract vascularization, so it can be reasonably assumed that in each particular transplant, the degree of bone formation reflects and is supported by the corresponding degree of vascularization. Our findings also suggest that transplant strength represents the result of a complex interplay between new bone formation and scaffold resorption.
Our results suggest that optimization of scaffold geometry, by varying lamellar spacing as we did here, or by varying other parameters such as HA/TCP ratios or surface roughness, may prove sufficient by itself to promote bone formation in the interior of moderately sized rigid scaffolds. They also begin to offer us insights into the optimal design of large-volume, clinically sized scaffolds.