Introduction
Bone can be damaged by trauma or disease and often bone graft substitutes are then needed for repair. Substitute bone can be derived from the patient (autograft) or from a donor (allograft). The common treatment is to use autologous bone grafts but this method has its drawbacks. It causes the generation of a second surgical site with increased donor site morbidity. Secondly, availability of autologous bone is limited [
1]. With the other option, using allograft material, there are risks of immune reaction and disease transmission [
2]. For this reason, there is a huge interest in developing new strategies for bone replacement. Marrow derived progenitor cells of adults represent a promising source of therapeutic tool and are known to differentiate along various mesenchymal lineages. The use of adult bone marrow stromal cells (MSCs) to achieve bone and cartilage formation and repair have met with less success and more problems than expected [
1]. In relation to bone formation, one of the largest problems has been nutrient delivery and waste removal associated with a lack of vasculature in implanted tissues leading to core necrosis and implant failure. It is clear that vascularisation is a critical consideration for any regenerative medicine approach [
3,
4]. Cartilage is an avascular tissue and, hence, does not suffer from this problem. However, regenerative medicine approaches to cartilage regeneration have also met with problems [
5], mainly because of the tendency of MSCs to naturally progress from forming stable, collagen type II expressing, cartilage to a more hypertophic phenotype characterised by expression of collagen type X.
In a recent paper we hypothesised that the natural tendency of chondrogenically primed MSCs to become hypertrophic might be a very desirable trait for bone tissue engineering applications [
6]. MSCs have been shown to progress along similar stages of endochondral ossification as observed during development [
7]. Recent successes in the induction of endochondral ossification from embryonic stem cells and murine bone marrow cells supported the feasibility of such an approach [
8‐
10]. There are several rationales behind the hypothesis that this route of bone formation would be more successful than intramembranous ossification. Firstly, chondrocytes normally reside in an avascular tissue and as a result are "designed" to function in a low oxygen environment, similar to what they would encounter upon implantation into an unvascularised region [
11]. Secondly, as stated, MSCs under
in-vitro conditions (almost) always become hypertrophic when cultured chondrogenically, the next step in the endochondral ossification pathway [
7,
12]. Thirdly, the release of factors from primed chondrogenic cells progressing along the endochondral route would be much more complex and controlled spatiotemporally than any growth factor combination we could devise in order to improve
in-vivo vascularisation and bone formation. Previously [
6], we demonstrated that chondrogenically primed MSC seeded scaffolds did indeed survive 4 weeks
in-vivo without core necrosis as evaluated histologically. Furthermore, we observed blood vessels in the chondrogenic samples only and data suggested that this was due to release of VEGF from these constructs as measured
in-vitro in chondrogenic pellets. However, we did not observe bone formation in any of the samples
in-vivo after 4 weeks. We hypothesised that either samples were not primed for long enough
in-vitro or they were not maintained
in-vivo for long enough to allow the process to occur.
In the current experiment we cultured MSC seeded scaffolds for a longer period
in-vitro to allow cells to migrate into the scaffolds prior to priming and to potentially form more matrix prior to implantation. In addition, samples were maintained
in-vivo for a minimum of 8 weeks. Our aim was to answer 3 specific questions. Firstly can adult human MSC seeded scaffolds undergo endochondral ossification
in-vivo to form bone for the purposes of bone repair/replacement? Secondly, can this process be further optimised by allowing mineralisation to occur
in-vitro for a brief period of time before implantation, thereby speeding up or enhancing the quantity of bone formed. Thirdly, what is the role of the donor and host cells in the process of endochondral ossification? To answer this final question we used a transgenic rat model ubiquitously expressing human placental alkaline phosphatase (hPLAP) as a recipient of wild type cells [
13,
14].
Materials and methods
human bone marrow cell culture
Bone marrow aspirates were obtained from three donors, 47, 57 and 69 years of age undergoing total hip arthroplasty after informed consent with approval of the local medical ethical committee (METC2004-142). The aspirates were plated as previously described [
6].
To create a pellet, suspensions of 200,000 cells per 15 ml tube were centrifuged at 200 g for 8 minutes. For the scaffolds, suspensions of detached cells were seeded with 1*10
6 cells per scaffold, divided into 500,000 cells in 100 μl on each side of the Collagen-GAG scaffolds as described previously [
15]. The constructs were cultured for 7 days in medium as used for expansion (control medium). Afterwards all samples were either maintained in control medium or replaced with chondrogenic or osteogenic medium for 28 days. Half of the medium was replaced every 3 days.
Chondrogenic medium consisted of high-glucose DMEM containing 50 mg/mL of gentamicin and 1.5 mg/mL of Fungizone (Invitrogen) 25 μg/ml L-ascorbic acid 2-phosphate, 100 mM of sodium pyruvate (Invitrogen), 1:100 insulin-transferrin-selenium (ITS; BD Biosciences, Bedford, MA), 10 ng/mL of transforming growth factor beta-2 (TGF-b2), (R&D Systems, Abingdon, United Kingdom) and 100 nM dexamethasone (Sigma, St. Louis, MO). The osteogenic medium consisted DMEM containing 10% fetal calf serum (Gibco, selected batch), gentamicin and 1.5 mg/mL of Fungizone (Invitrogen) 0.1 mM L-ascorbic acid 2-phosphate, 10 mM beta-glycerol phosphate, 100 nM dexamethasone.
To investigate if bone formation in vivo can be enhanced by allowing mineralisation to occur before implantation, we have applied chondrogenic medium for 21 days and then switched to mineralizing medium conditions for the last 7 days of culture. For the switch 1 condition the chondrogenic medium was replaced after 21 days of culture with osteogenic medium for the remaining period of 7 days. For the switch 2 condition after 21 days of culture in chondrogenic medium, 10 mM beta-glycerol phosphate (as a source of phosphate to allow for mineralization) was added to the chondrogenic medium for the remaining period of 7 days.
Gene expression analysis
To confirm chondrogenic potential of MSCs prior to implantation, gene expression analysis of GAPDH, Sox9, cbfa1, collagen type II and collagen type X was performed as described previously [
16] In addition, samples cultured as pellets were harvested from each MSC donor, fixed in 4% phosphate buffered formalin and embedded in paraffin for collagen type II immunohistochemistry (II-II6B3 antibody, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, under contract N01-HD-6-2915 from the National Institute of Child Health and Human Development).
In vivo implantation of hMSC
To evaluate bone formation, cultured constructs were implanted subcutaneously in athymic mice (Balb/C nudes, CDL Nijmegen). For each donor, 3 constructs of each condition were implanted. Before surgery, the skin on both lateral sites of the spine was cleaned with 70% alcohol and 4 subcutaneous pockets were created in each mouse. The tissue engineered samples or pellets were inserted and the pockets closed. Three empty scaffolds were also implanted. Two of these were maintained for the duration of the culture period in expansion medium and one of these in chondrogenic medium. Eight and fourteen weeks after surgery, the animals were euthanized by CO2. The explanted samples were fixed in 4% paraformaldehyde, decalcified in formic acid and embedded in paraffin. The experiments were approved by the Dutch animal experiment committee.
Micro CT imaging
All samples were scanned using micro-CT (Skyscan model 1072, Kontich, Belgium) with a source of 50 kV/98mA without using a filter (resolution 8.1 μm per pixel). Each sample was rotated 180 degrees with a rotation step of 0.90 degrees, exposure time 2.9 seconds. 3D reconstruction, analysis and visualizations were made with NRecon version 1.6, CT-analyzer V1.9 (Skyscan) and 3D-Doctor™ (Able Software Corp., Lexington, United States).
Histomorphometrical analysis
Sections were stained with haematoxylin-eosin and evaluated for presence or absence of bone. A Fisher exact test was used to evaluate statistical significance. Histomorphometry was performed on 2-4 sections of each sample. From each section, low magnification digital images were made, images were pseudo colored and measurements were performed with image analyses techniques (Leica Qwin Pro-image analysis system, Wetzlar, Germany) to obtain the percentage of bone, bone marrow and other tissue.
Rat MSC isolation, culture and implantation
MSCs from 5 month-old inbred wild-type Fischer 344 (F344) rats were isolated and cultured according to standard procedures as described elsewhere [
15]. Culture and scaffold seeding was performed exactly as for the human MSCs as described above. The second switch condition was employed for the rat component of this study. Following 5 weeks
in vitro, three constructs (scaffolds) of each condition were implanted subcutaneously into immunocompetent co-isogenic hPLAP-transgenic (human Placental Alkaline Phosphatase) F344 rats for 8 weeks. Animals were sacrificed by exsanguination under ketamine/xylazine anesthesia. Scaffolds were harvested and fixed in 40% ethanol at 4°C for 48 h, dehydrated and embedded in modified methylmetacrylate [
17].
Immunohistochemistry for collagen type II
To analyze collagen type II expression, sections were incubated with 0.1% pronase (Sigma, St Louis, MO) for antigen retrieval and 1% hyaluronidase (Sigma, St Louis, MO). Sections were incubated for 2 h at room temperature with mouse monoclonal antibody against collagen type II (II-II6B3 antibody, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, under contract N01-HD-6-2915 from the NICHD).
hPLAP immunohistochemical staining
For histochemical staining of the marker enzyme hPLAP deplastisized sections were rehydrated and heated at 65°C for 30 min in deionized water to block endogenous alkaline phosphatase activity. Cells expressing hPLAP were histochemically stained by incubation with an AP substrate (TRIS-HCl buffer (0.2 M, pH 8.5) containing Naphtol AS-MX 0.3 mg/ml (Sigma) and New Fuchsin 0.1 mg/ml (Chroma)) at room temperature for 1 hour and counterstained with haematoxylin.
Discussion
Tissue engineering approaches to bone repair have thus far been disappointing. Recent interest has focused on the process of endochondral ossification as a possible means to generate bone for regenerative medicine purposes [
6,
8,
18‐
20]. The release profile of factors that occurs during endochondral ossification is complex and coordinates the formation of bone from a cartilage template [
21]. Here we show that chondrogenically differentiated adult human and rat MSCs seeded into collagen GAG scaffolds give rise to bone formation via endochondral ossification
in-vivo. Previously, it was demonstrated that this was possible with murine embryonic stem cells [
8] as well as murine adult bone marrow stromal cells [
9,
10]. The data from our study are also supported by the recent publication by Chan
et al[
22] demonstrating that endochondral ossification is required for haematopoietic stem cell niche formation with a subpopulation of foetal progenitor cells giving rise to bone with a marrow cavity only if they would normally undergo endochondral ossification as opposed to intramembranous ossification. Even more recently, Janicki
et al[
23] demonstrated the same mechanism of bone formation via endochondral ossification using human MSCs and β-tricalcium phosphate with a 6 week
in-vitro chondrogenic pre-culture.
Initial results presented in this manuscript demonstrate that eight weeks of implantation was not sufficient to ossify the complete construct. In case of a completely cartilaginous construct (such as the pellets we used) the remaining tissue is cartilage and the sample harvested after 14 weeks demonstrated that the complete construct is subsequently turned into bone and bone marrow. The bone is then only localised at the outer rim, probably due to a lack of mechanical stimulation that is prerequisite for bone maintenance. The technical problems associated with homogenous cell seeding will likely become relevant when upscaling the procedure towards application in patients. Use of bioreactors to improve cell seeding efficiency and also mechanical integrity could be considered [
24‐
26].
Addition of β-glycerophosphate
An important consideration in the generation of bone via endochondral ossification is the optimum differentiation stage at which one can implant. Ideally, the further along the differentiation pathway a construct is prior to implantation the faster it would fulfil its role
in-vivo. To assess this we cultured scaffolds in both osteogenic medium as a negative control of bone formation and in chondrogenic medium for 3 weeks with a switch to standard osteogenic medium for one week (switch 1) to begin the osteogenic differentiation process. Despite the brief period of exposure to these culture conditions, no bone formation was observed
in-vivo. We hypothesised this was due to a lack of vascularisation due to reduced release of pro-angiogenic factors that we had previously observed
in-vitro[
6]. However upon close inspection, blood vessels were observed in all 4 culture conditions. As a further evaluation of the effect of the presence of mineralisation before implantation, we simply added β-glycerophosphate to the chondrogenic media for 1 week which we had also shown previously to cause mineralisation. Unfortunately only a single pellet was retrieved in the human MSC pellets as the other two could not be located. However, bone formation under this switch 2 (glycerophosphate only) condition was also observed in 3 out of 3 scaffolds retrieved in the rat study (Data not shown). Thus we are confident that addition of glycerophosphate for a week will not prevent endochondral ossification as we observed in the switch 1 (full osteogenic medium switch). These findings would suggest that pre-mineralisation alone will not prevent the process of endochondral ossification occurring
in-vivo. It is likely another factor in the osteogenic medium that prevents bone formation
in-vivo, possibly the presence of serum. The lack of retrieved samples in the human MSC study can not be ignored however as it is possible that these samples could not be retrieved because they were resorbed by the host. Pre-mineralisation might offer the advantage of stiffer scaffolds upon implantation which would greatly improve the options in load bearing situations and ideally reduce the time required for internal/external fixation.
The role of host and donor in endochondral ossification
Determining the origin of host and donor cells in this type of experiment is a difficult task. Here we used transgenic rats expressing hPLAP into which we implanted scaffolds containing wild type cells of the same inbred strain. This approach has two goals. Firstly, to determine the origin of the bone forming cells in the process of endochondral ossification and secondly to confirm that the results observed in immunocompromised mice could be reproduced in immunocompetent animals. The utility of this approach for the identification of donor/host cells in a variety of tissues has been demonstrated previously [
13,
14]. In accordance with the experiments using human MSCs in immunocompromised mice, bone formation occurred only under chondrogenic and β-glycerophosphate conditions in rats. Analysis of hPLAP expression in the various tissues clearly demonstrated the presence of host and donor-derived cells. Embedded in the bone matrix, positively and negatively staining cells were observed, suggestive of the presence of cells of both host and donor origin, indicating that at least at earlier time points the donor cells are actively involved in the formation of bone. In order to clearly identify the roles of both host and donor cells, a timecourse analysis coupled with the reverse scenario (Transgenic cells into wild type animals) should be performed. Eight weeks after implantation all osteoblasts and lining cells were of host origin, suggesting that the bone formed from that time point on will be host derived.
Conclusions
The work presented in this article suggests that the induction of chondrogenesis
in vitro vs osteogenesis offers an improved approach to bone repair and regeneration
in vivo. As discussed in a previous article [
6], we believe this is in part due to the paracrine effects of these cells with different release profiles of important factors such as VEGF, MMPs and other growth factors at critical stages in the process. It is clear from this work that chondrogenic priming of cells, particularly of adult human MSCs offers an extremely promising route to bone formation and repair that will undoubtedly be pursued in the coming years as an alternative to the standard intramembranous ossification approach of tissue engineering bone.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EF was involved in study conception and design, cell culture, scaffold seeding in Rotterdam and Vienna and all aspects of analysis and manuscript preparation. SB was involved in study design, performed in vivo nude mouse study, analysis of all experimental data and paper preparation. KO performed hPLAP component of study including cell preparation, scaffold seeding and animal surgeries and subsequent hPLAP histochemical analysis. WK was involved in study design, MSC isolation and culture and PCR analysis, NK was involved in all aspects of histological and histochemical analysis, FOB was responsible for scaffold design and fabrication, RBJ was involved in study conception and article preparation, JV was involved in study conception and article preparation, VC performed micro ct analysis and was also involved in histomorphometric analysis, JJ was involved in study conception and article preparation, RE was involved in study conception and design specifically related to hPLAP animal model and article preparation, GvO was involved in study conception and design, data analysis and article preparation. All authors read and approved the final manuscript.