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J Dent Res 85(7):633-637, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Transplanted Endothelial Cells Enhance Orthotopic Bone Regeneration

D. Kaigler1, P.H. Krebsbach2, Z. Wang2, E.R. West3, K. Horger3, and D.J. Mooney4,*

1 Depts. of Periodontics/Prevention/Geriatrics,
2 Biologic and Materials Sciences, and
3 Chemical and Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; and
4 Div. of Engineering and Applied Sciences, Harvard University, 29 Oxford St., 325 Pierce Hall, Cambridge, MA 02138, USA

* corresponding author, mooneyd{at}deas.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to determine if endothelial cells could enhance bone marrow stromal-cell-mediated bone regeneration in an osseous defect. Using poly-lactide-co-glycolide scaffolds as cell carriers, we transplanted bone marrow stromal cells alone or with endothelial cells into 8.5-mm calvarial defects created in nude rats. Histological analyses of blood vessel and bone formation were performed, and microcomputed tomography (µCT) was used to assess mineralized bone matrix. Though the magnitude of the angiogenic response between groups was the same, µCT analysis revealed earlier mineralization of bone in the co-transplantation condition. Ultimately, there was a significant increase (40%) in bone formation in the co-transplantation group (33 ± 2%), compared with the transplantation of bone marrow stromal cells alone (23 ± 3%). Analysis of these data demonstrates that, in an orthotopic site, transplanted endothelial cells can influence the bone-regenerative capacity of bone marrow stromal cells.

KEY WORDS: bone marrow stromal cells • endothelial cells • angiogenesis • osteogenesis • tissue engineering


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In dentistry, the field of tissue engineering will likely have great impact in the area of bone regeneration. Cell transplantation is a widely used tissue-engineering strategy, and can be used to examine bone development and regeneration from a variety of cell types. Multipotent cells derived from bone marrow are fast becoming a popular choice as a source of osteoprogenitor cells for the tissue engineering of bone (Ishaug-Riley et al., 1997; Gao et al., 2001). In cell cultures generated from suspensions of marrow, colonies form from a precursor cell termed the CFU-F (Friedenstein et al., 1978; Latsinik et al., 1986), and the progeny of the CFU-F are what have been defined as bone marrow stromal cells. These cells are capable of extensive proliferation and differentiation into several phenotypes, including bone, cartilage, fibrous tissue, adipose tissue, and hematopoiesis-supporting reticular stroma (Krebsbach et al., 1999); additionally, they have potential applications in tissue repair strategies (Krebsbach et al., 1997).

It is well-established that bone formation is an angiogenesis-dependent process (Gerber et al., 1999), and endothelial cells have long been known for their role in forming blood vessels that supply oxygen and nutrients to developing bone tissue. However, it has been suggested, more recently, that endothelial cells may play a more direct role in bone development and formation, through their interactions with osteoprogenitor cells (Villars et al., 2002) and, under certain conditions, their production of specific bone-inductive factors (Bouletreau et al., 2002).

This study addresses the hypothesis that endothelial cells can modulate the osteogenic potential of bone marrow stromal cells in an orthotopic bone regeneration model. The aim of this study was to determine if, in a critical-sized calvarial defect model, bone marrow stromal cells co-transplanted with endothelial cells on biodegradable poly-lactide-co-glycolide (PLGA) scaffolds produce more bone than bone marrow stromal cells transplanted alone. Several studies have used ectopic bone formation models to determine the osteogenic potential of these cells (Krebsbach et al., 1997; Bruder et al., 1998), and we have previously demonstrated that co-transplantation of endothelial cells and bone marrow stromal cells enhances bone regeneration in this type of model (Kaigler et al., 2005). Although fundamental questions can be addressed in these types of systems, the orthotopic environment is clearly distinct, and orthotopic bone models (Bidic et al., 2003; Blum et al., 2003; Akita et al., 2004) are more clinically relevant.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
PLGA Scaffold Fabrication
A copolymer of D,L-lactide and glycolide (85:15 molar ratio) (Alkermes, Cambridge, MA, USA) was utilized in a gas-foaming process (Mooney et al., 1996) to form scaffolds for cell transplantation. A detailed description of the protocol is found in Appendix 1.

Cell Culture
Human bone marrow was collected from patients undergoing iliac bone graft procedures (with University of Michigan IRB approval and informed patient consent), and bone marrow stromal cells were isolated as previously described (Krebsbach et al., 1997).

Human dermal microvascular endothelial cells were purchased (Cambrex BioScience Walkersville, Inc., Walkersville, MD, USA) and cultured in EGM-2 MV containing 5% fetal bovine serum (FBS) (Cambrex BioScience). When cells reached 85–90% confluence, they were subcultured, and cells were used at their 5th passage in culture.

Before cells were seeded, scaffolds (8.5 mm diameter, 2 mm thickness) were immersed in 100% ethanol for 1 hr, followed by 5 rinses of phosphate-buffered saline. We used 2 x 2 gauze to "wick" polymers (Johnson & Johnson, New Brunswick, NJ, USA) for the removal of residual saline just prior to cell-seeding, and 50-µL cell suspensions (1.0 x 106 cells) were then added to each scaffold, dropwise, with a pipette. Scaffolds were then left at 37°C for 1 hr before implantation. A more detailed protocol for cell culture can be found in Appendix 1.

Critical-sized Defect Model
PLGA scaffolds alone, seeded with bone marrow stromal cells, or seeded with both bone marrow stromal cells and endothelial cells, were prepared for transplantation. A trephine bur was used to create a circular 8.5-mm-diameter osteotomy in the rat cranium (rat cranium critical-sized defect = 8 mm) (Schmitz and Hollinger, 1986) of nude rats, producing the critical-sized defect. The detailed protocol can be found in Appendix 1.

Histologic Techniques
Paraffin-embedded matrices, after being harvested and fixed, were cut into serial sections and placed on glass slides for histological analysis, with the use of hematoxylin and eosin (H & E) staining and CD31 immunohistochemical staining. A detailed protocol can be found in Appendix 1.

Blood Vessel and Bone Analysis
Blood vessels present in the implants were analyzed for their total number. Blood vessels were identified in H & E-stained tissues as previously described (Kaigler et al., 2005). To allow for the identification and counting of blood vessels formed from transplanted human cells, matrices from experimental conditions that included transplanted endothelial cells were stained for the presence of human CD31 antigen. Micro-computed tomographic images (µCT MS8X-130; EVS Corp., Toronto, ON, Canada) and H & E-stained sections were analyzed for quantitative and qualitative histomorphometric differences in bone formation between the conditions. For a detailed description of the blood vessel and bone analysis, see Appendix 1.

Statistical Analysis
Six scaffolds were prepared, implanted, and analyzed per condition and time-point, and statistical analysis was performed with the use of Instat software (GraphPad Software, San Diego, CA, USA). All data were plotted as mean ± standard error of the mean (SEM), unless otherwise noted. Statistically significant differences in histomorphometric analysis were determined by two-tailed Student t tests, and statistical significance was defined as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first examined the defects 6 wks after scaffold implantation, to determine if transplanted endothelial cells contributed to defect neovascularization. Blood vessel densities within the defects were determined, and the total numbers of vessels formed between both conditions were similar (Fig. 1aGo). However, in the co-transplantation condition, 5% of the total functional vessels formed were human-derived, as determined by human CD31(+) immunostaining of vessels (Fig. 1bGo). In contrast, in the control group with no endothelial cells, no human-cell-derived vessels were present. Samples were next analyzed at 12 wks, and the nature of the angiogenic response between conditions was determined. The densities of total blood vessels in defects were higher in both experimental groups at 12 wks, as compared with those at 6 wks, but there was no statistical difference between transplants of bone marrow stromal cells and endothelial cells and transplants of bone marrow stromal cells alone (Fig. 1cGo). As a control for the neovascular effect of the bone marrow stromal cells, a third group of polymer scaffolds (poly-lactide-co-glycolide) that did not contain any cells was included at the 12-week time-point. In this group, the total angiogenic response was significantly lower (p < 0.05) than in the other two groups at this time-point (Fig. 1cGo). Quantification of human-derived blood vessels revealed that 2% of the total functional vessels formed in the co-transplantation group were derived from transplanted cells, as determined by human CD31(+) immunostaining of vessels (Fig. 1dGo). In contrast, in the control groups with no endothelial cells, no human-cell-derived vessels were present. The actual summary values and standard deviations for these values are presented in Appendix 2 (Table 1).


Figure 1
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  		Figure 1. Blood vessel analysis. (a) Bone marrow stromal cells were transplanted with (BMSC/EC) and without endothelial cells (BMSC). Six weeks following transplantation, the total numbers of blood vessels were counted and normalized per unit area following retrieval of implants. (b) Human-derived vessels were determined by human CD31(+) staining of vessels and plotted as percentages of total functional vessels. (c) 12 weeks following transplantation, total blood vessels were counted in both conditions, and an additional group, control implant condition of poly-lactide-co-glycolide scaffolds that did not contain any cells (PLGA), was also included at this time-point. (d) Human-derived vessels were determined by CD31(+) stained vessels and expressed as percentages of total vessels. Values represent mean ± standard error of the means (unless otherwise noted). *Statistically significant difference (p < 0.05), as compared with BMSC condition; **statistically significant difference as compared with both of the other experimental conditions (n = 6).

 
Transplanted endothelial cells were present at 6 and 12 wks, and it was next determined whether the presence of these cells affected bone regeneration at these time-points. Qualitative histological analysis of samples from both conditions, harvested at 6 wks, suggested the presence of bone formation, as determined by the organization of the collagen matrix and the presence of osteocytes within lacunae in focal regions. This tissue had a wavy, trabecular appearance, with little to no presence of hematopoietic tissue formation (Figs. 2a, 2bGo). Quantitative µCT analysis of samples at this time-point confirmed the presence of a mineralized extracellular matrix, indicative of bone, and showed that although the relative amounts of total bone formed in both conditions were the same (Fig. 2cGo), there was a statistically significant (p < 0.05) increase in the BMD of bone formed from co-transplants (320 ± 30 mg/cm3), compared with the BMD of bone formed from bone marrow stromal cell transplants (240 ± 30 mg/cm3) (Fig. 2dGo).


Figure 2
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  		Figure 2. Bone analysis at 6 wks. Photomicrographs of hematoxylin & eosin (H & E)-stained sections retrieved 6 wks after transplantation of (a) bone marrow stromal cells alone (BMSC) or (b) with endothelial cells (BMSC/EC). (c) Following retrieval of the scaffolds used to transplant the cells, total bone volume was quantified at this time-point by µCT. (d) Bone mineral density was also quantified. Values represent mean ± standard error of the means (unless otherwise noted). *Statistically significant difference (p < 0.05), as compared with BMSC condition. Size bars are shown in (a) and (b) (n = 6).

 
Evaluation of later-stage bone tissue formation was performed 12 wks following the transplantation of scaffolds. Gross histological and µCT analyses spanning the entire defect revealed large bone foci following transplantation of bone marrow stromal cells and co-transplantation of bone marrow stromal cells with endothelial cells (Fig. 3Go). However, regenerated bone tissue nearly bridging the calvarial defects was identified histologically only in the condition where both bone marrow stromal cells and endothelial cells were co-transplanted (Fig. 3cGo). Micro-CT analysis confirmed the presence of mineralized matrix in these areas (Fig. 3dGo). High-magnification histological examination of samples revealed larger foci of bone (Fig. 4aGo) relative to the smaller bone foci observed at 6 wks. More organized areas of matrix were surrounded by less organized regions of bone trabeculae, and these regions were devoid of marrow or hematopoietic tissue. Quantitative µCT analysis of bone at this time-point showed that the BMD of bone formed was not statistically different between the two conditions (Fig. 4bGo). The BMD of the native bone of the rat calvarium (periphery of defect) is included (Appendix 2-Table 2) as a base standard for the assessment of the mineralized matrix of the regenerated bone relative to the native bone. Finally, in the co-transplant condition, a statistically significant (p < 0.05) increase (40%) in total bone volume fraction (33 ± 2%) was found, as compared with that in transplants of only bone marrow stromal cells (23 ± 3%) (Fig. 4cGo). The additional control group (scaffold containing no cells) resulted in minimal bone regeneration, since the total bone volume was only 6%, and this was significantly lower than in both of the conditions where bone marrow stromal cell transplantation was used.


Figure 3
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  		Figure 3. Calvarial defects at 12 wks. (a,c) Photomicrographs (H & E-stained) and (b,d) µCT images of cross-sections of the calvarial defect sites implanted with (a,b) bone marrow stromal cells alone and (c,d) co-transplantated with endothelial cells. Arrows demarcate defect margins (8.5-mm defect). Size bars are shown in (a) and (c).

 

Figure 4
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  		Figure 4. Bone formation at 12 wks. (a) Photomicrograph of an H & E-stained section from a co-transplant sample retrieved at 12 wks. Micro-CT analyses of (b) bone mineral density from bone marrow stromal cell transplants (BMSC) and bone marrow stromal cell, endothelial cell co-transplants (BMSC/EC). (c) Total bone volume fraction in BMSC transplants and co-transplants of BMSC and EC. An additional experimental group of scaffolds alone with no cells (PLGA) was included at this time-point. A scale bar is shown in (a). Values represent mean ± standard error of the mean. *Statistically significant difference (p < 0.05), as compared with BMSC condition (n = 6).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tissue-engineering approaches to the treatment of craniofacial defects are dependent on several key factors: an osteoprogenitor cell type, an appropriate osteoinductive signal, and a sufficient angiogenic response. In particular, the use of a cell transplantation approach combining different types of scaffolds with osteogenic cells has been widely studied (Ohgushi et al., 1990; Yaszemski et al., 1996); however, a key limitation of bone formation studies in ectopic sites is that the microenvironment does not inherently contain osteoinductive factors to support bone regeneration. In an orthotopic site (calvarial defect), components of the microenvironment can be osteoconductive (Mehrara et al., 1999; Greenwald et al., 2000), creating greater potential for bone regeneration. For this reason, we used a critical-sized osseous defect model in the current study, to examine the effects of endothelial cells on bone formation by transplanted bone marrow stromal cells.

Transplanted endothelial cells have the ability to form blood vessels (Schechner et al., 2000; Nor et al., 2001; Peters et al., 2002) and could potentially increase neovascularization to regenerating tissues. In the present study, transplanted endothelial cells were present in the vasculature up to 12 wks after transplantation; yet their contribution was not sufficient to increase the overall angiogenic response, as compared with the condition in which bone marrow stromal cells were transplanted alone. Both transplantation conditions showed statistically significant increases in the angiogenic response, compared with the negative control (scaffold implanted with no cells). These findings could be due to a maximal angiogenic response from the bone marrow stromal cells’ production of VEGF (Fuchs et al., 2001; Kaigler et al., 2003).

Another finding to note was that blood vessel formation derived from the endothelial cells was not as dramatic as reported in other studies that have used transplantation of endothelial cells in different systems (Schechner et al., 2000; Nor et al., 2001). This may have been caused by the lower density of endothelial cells used in our studies (4–5 times lower than in other studies), or by the lack, in this study, of exogenous growth factors to augment the angiogenic response elicited by transplanted endothelial cells. Finally, most previous studies examined blood vessel formation over a short time interval (days to a couple of weeks). In our studies, we examined blood vessel formation at later time intervals (6 and 12 wks), to evaluate bone regeneration, and while human endothelial cells were detected at these time-points, they were no longer present in high numbers. However, the transplanted endothelial cells may serve to organize and assist in establishing an initial angiogenic response, which can then be sustained by endothelial cells from the host organism.

Transplanting bone marrow stromal cells, alone or with endothelial cells, led to focal areas of bone formation at 6 wks. There was no statistically significant difference in total bone volume between these two conditions, but BMD of bone tissue formed at this time-point was statistically higher in the co-transplant condition. One likely explanation for this finding is that endothelial cells directly regulate osteogenic differentiation of bone marrow stromal cells in vivo, as has been shown in vitro (Villanueva and Nimni, 1990; Collin-Osdoby, 1994; Villars et al., 2002; Kaigler et al., 2005). The co-transplantation of endothelial cells with bone marrow stromal cells provides an environment in which the two cell types are close to one another. This presumably allows transplanted endothelial cells to interact with bone marrow stromal cells earlier than host-derived endothelial cells, which would first need to migrate to and infiltrate the scaffold. Thus, signaling from the transplanted endothelial cells could have resulted in more rapid mineralization of bone matrix, as measured by the BMD.

Examination of bone formation at 12 wks revealed a significant effect of endothelial cell transplantation on total bone formation. BMD of bone formed in both conditions was statistically the same at this point, yet there was an overall greater amount of bone formed in the co-transplantation condition. A possible explanation for this finding is that the human bone marrow stromal cells could be more responsive to the transplanted human endothelial cells than the endothelial cells of the host. Yet, the findings of this study are consistent with the results of a previous study which demonstrated that co-transplantation of endothelial cells increased the bone-forming capacity of bone marrow stromal cells in an ectopic site (Kaigler et al., 2005). Clearly, further studies need to be conducted for better elucidation of the actual mechanisms underlying these findings.

In summary, these studies demonstrate that transplanted endothelial cells can enhance the bone-forming capacity of bone marrow stromal cells in an orthotopic critical-sized defect model. Endothelial cells influenced bone formation in terms of both the rate at which bone matrix was mineralized, and the total bone-regenerative capacity. The bone marrow serves as an excellent source of cells with osteogenic potential, and new methods have now emerged that enable endothelial cells and their progenitors to be isolated from bone marrow (Shi et al., 1998; Jackson et al., 2001; Reyes et al., 2002). From a therapeutic standpoint, it would be convenient and efficient if these two cell types could be isolated from the same biopsy, expanded ex vivo, and re-transplanted to manage craniofacial bone defects.


   ACKNOWLEDGMENTS
 
We thank John Baker and Chris Strayhorn for providing histology support. The authors are grateful to the NIDCR for funding an Individual Pre-doctoral Dental Scientist Fellowship (F30 DE05747) to DK, and for research funding to the laboratories of PK (RO1-DE013835). This work was supported by the U.S. Army Research Laboratory and the U.S. Army Research Office under grant number DAAD190310168.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received July 7, 2005; Last revision March 21, 2006; Accepted April 9, 2006


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