Significant research efforts have been undertaken during the last decades to treat musculoskeletal disorders and improve patient’s mobility and quality of life. The goal is the return of function as quickly and completely as possible. Cellular therapy has been increasingly employed in this setting. The design of this study was focused on cell-based alternatives. The present study aimed at investigating the bone regeneration capacity of xenogeneic human bone marrow-derived mesenchymal stromal cell (hMSC) implantation with tricalcium phosphate (TCP) granules in an immunocompetent rabbit model of critical-size bone defects at the femoral condyles. Two experimental groups, TCP and hMSC + TCP, were compared. Combination of TCP and hMSC did not affect cell viability or osteogenic differentiation. We also observed significantly higher bone regeneration in vivo in the hMSC + TCP group, which also displayed better TCP osteointegration. Also, evidence of hMSC contribution to a better TCP osteointegration was noticed. Finally, no inflammatory reaction was detected, besides the xenotransplantation of human cells into an immunocompetent recipient. In summary, hMSC combined with TCP granules is a potential combination for bone regeneration purposes that provides better preclinical results compared to TCP alone.
Despite the numerous advances in orthopaedic surgical techniques and new biomaterials, the repair of bone lesions continues to have a great room for improvement. Furthermore, the risk of bone diseases is far more prevalent due to aging. Bone fracture repairs have been intensively investigated at both clinical and basic level and still 5–10% of fractures resulted in either delayed or no repair [
The possibility of repairing an injured tissue by regeneration seems to be an attractive therapeutic option. Bone tissue remodelling process provides the capacity of self-regeneration after injury and the continual adaptation of bone mass and its architecture to the mechanical load [
Calcium phosphates have been widely studied and used for bone repair [
Regarding combination of calcium phosphates with cellular components, bone marrow mesenchymal stromal cells (MSC) became well known at the end of the 1990s due to the evidence of being capable of multilineage differentiation. This property favored their use in bone tissue engineering, mostly in combination with an osteconductive scaffold as a graft material [
Previous studies have highlighted that autologous bone marrow stromal cells (MSC) are capable of regenerating bone defects when used in combination with bone substitutes [
The main purpose of the study was to probe the immunoprivileged properties of hMSC in a xenogeneic setting. We attempted to investigate the bone regeneration capacity of the xenograft in a critical-sized bone defect in an immunocompetent rabbit recipient. The challenge of the study was to get the viable addition of hMSC embedded in a common synthetic scaffold to promote bone regeneration in a xenogeneic model. A positive result could have a clinical relevance for any orthopaedic procedure requiring bone formation and may serve as preclinical basis to support the use of allogeneic cells.
Iliac crest bone marrow aspirates (5 ml) were obtained from patients that underwent spinal fusion for degenerative disc disease. They were otherwise healthy, and all of them were subjected to clinical and analytical evaluation to exclude the presence of relevant diseases and they were not receiving medical treatment for any condition, other than analgesics for the spinal degenerative disease. Median age of the donors was 60 years (range: 28–80 years), and male/female ratio was 1. Specimens were harvested according to the tenets of the Declaration of Helsinki and the Ethical Committee of the Hospital Universitario de Salamanca. All donors provided informed consent for the bone marrow sampling. A mononuclear fraction of bone marrow (CMN) was isolated by density-gradient centrifugation. Briefly, the bone marrow aspirate was diluted in Hank’s balanced salt solution to increase the volume up to 12 ml. This cell solution was transferred to a centrifuge tube with 4 ml of Ficoll-Hypaque (Biochrom KG, Berlin, Germany) and was centrifuged at 1500 rpm for 30 min at room temperature. The interface cell layer was washed twice with Hanks 10 min at 1200 rpm at room temperature. The pellet was suspended with DMEM medium (Gibco BRL, Pailey, United Kingdom). A concentration of 106 CMN/cm2 mononuclear isolated cells were seeded in a dish (T75 flaks) and cultured with DMEM supplemented with 10% fetal bovine serum (SBF; Bio Whittaker, Belgium) and antibiotics and incubated at 37°C with 5% CO2 in a humidified atmosphere. At 2-day intervals, the medium was replaced, and thus nonadherent cells were removed. Cells were allowed to expand up to reach around 70% of confluence. Then they were trypsinized and further subcultured at a density of 2.5 × 103 cells/cm2. Cells were maintained until the 3rd passage, with a median of 11.48 ± 1.02 days in culture. At this stage, all the immunophenotypic analysis, the multilineage differentiation studies, and the remaining experiments were performed.
Cell culture was characterized by flow cytometric analysis (FCA) for specific surface antigens, including CD105, CD73, CD90, CD34, CD45, CD14, CD19, and HLA-DR, in accordance with the international Society for Cellular Therapy (ISCT) recommendations [
For osteogenic differentiation, the hMSC were cultured with specific differentiation medium NH OsteoDiff Medium (Miltenyi Biotec, Germany). The hMSC culture was changed every 3 days during 10 days [
For adipogenic differentiation, the hMSCs were cultured with differentation medium NH AdipoDiff Medium (Miltenyi Biotec, Germany). The hMSC culture was changed every 3 days for 21 days. Afterwards, the monolayer was washed with PBS, 10% formalin fixed for 2 min at room temperature, and then incubated for 1 hour with 1 ml Oil Red O solution (Merk, Darmstadt, Germany) at room temperature.
A synthetic porous ceramic graft material composed of tricalcium phosphate, commercially available as Conduit TCP (DePuy Orthopaedics Inc.) was employed as scaffold either alone or in combination with the hMSC. Conduit TCP consists of irregular granules with interconnected porosity of about 70% and pores of 1–600
All animal handling and surgical procedures were conducted according to the European Community guidelines for the care and use of laboratory animals (Directive 2010/63/EU) and approved also by the local ethical committee of the University of Salamanca, in accordance with Spanish law (RD 53/2013).
Fourteen immunocompetent mature male New Zealand rabbits weighing between 3.0 ± 0.5 kg were injected intramuscular an anaesthesia mixed of Xylacine 5 mg/kg (Rompun® 2%, 25 ml) and Ketamine 35 mg/kg (Ketolar® 50 mg/ml). Anesthetized sate was maintained with isoflurane and oxygen ventilation. Once each animal was anesthetized, the knees were disinfected with 4% chlorhexidine and shaved. Once the femoral condyle was exposed, an established bone critical-size defect [
Critical size femoral defect of 6 mm of diameter in a rabbit model.
The rabbit condyles were fixed in 10% neutral buffered formalin. The regions containing the defects were dehydrated in graded series of alcohol/water mixture followed by complete dehydration in absolute alcohol. Afterwards, the specimens were embedded in poly-methylmethacrylate resin and cut into 5–7
Assessment of bone regeneration was performed following a modification of the histological evaluation method from Lucaciu et al. [
Histological evaluation record.
Histological score |
---|
To identify the presence and participation of the hMSC in bone regeneration, immunofluorescence detection of a glucosylated protein present in the human mitochondrial membrane was detected by using a mouse monoclonal antibody (MAB1273 Millipore). Samples were analyzed and photographed under a photomicroscope (Zeiss Scope A1) equipped with epifluorescence and appropriate filter sets.
The histopathological results were scored in a double-blinded manner, and the figures presented in the manuscript are representative images.
Statistical analysis of the data was performed with the IBM SPSS (v. 23.0) application. Normal distribution of data from both, control and experimental groups, was tested with Shapiro-Wilk test (recommended for samples with
In all cases, hMSC were isolated and expanded in vitro and acquired the characteristic spindle-shape morphology. As indicated in the methods, cells were grown up to third passage and median time for each passage was 11.48 ± 1.02 days. After the flow cytometric analysis, the characteristic immunophenotypic profile was demonstrated. Cells were positive for CD90, CD73, and CD105 and negative for CD45, CD34, CD14, CD19, and HLA-DR (Figure
Immunophenotypic profile by flow cytometry.
In addition, multilineage differentiation into osteblasts and adipocytes was demonstrated by BCIP-NBT and Oil-Red-O staining, respectively (Figure
In vitro multilineage differentiation of hMSC: (a) control, (b) osteoblastic differentiation, and (c) adipocytic differentiation (scale bar: 50
There were no complications either during the surgical procedure, the postoperative course, or the bone biopsies performed 12 weeks after surgery. Only two of the cases presented infection signs in defects treated only with TCP, so these were discarded for the analysis.
Histology sections were blindly examined by bright-field microscopy. All samples showed signs of bone regeneration, in a greater or lesser extent, and no infiltration by inflammatory cells was found. However, bone formation was different between the two groups: TCP and hMSC-TCP. In particular, toluidine blue-stained sections from the TCP-only group revealed that the defect was still evident (Figure
Photomicrographs of histological sections stained with toluidine blue taken from the TCP group (a–c). (a) The defect area was still evident 12 weeks after surgery. Connective tissue and adipose bone marrow were generated at the injured area (scale bar: 1000
Photomicrograph of histological section from the TCP group. Evidence of empty spaces filled by adipose bone marrow due to the absence of osteointegration of TCP granules (scale bar: 150
Concerning the sections from the hMSC-TCP group, they showed an almost complete regeneration of the bone defect (Figure
Photomicrographs of histological sections stained with toluidine blue taken from the hMSC-TCP group (a–c). (a) Almost complete bone defect regeneration (scale bar: 1000
To identify and confirm that the implanted hMSC had survived and contributed to the regeneration process, the immunofluorescence detection of human mitochondrial antigen was performed (Figure
Fluorescent images of the TCP group (a) and the hMSC-TCP group (b) (scale bar: 150
After 12 weeks of implantation, in the TCP group, there was very scarce bone formation and it was mostly located at the periphery or at the surface, whereas in most of the hMSC-TCP-treated animals, bone formation was observed both in the central and in the peripheral regions of the lesion. Osteoblasts, osteocytes, and osteoclasts were scarcely observed in the TCP group, but they were abundantly observed in all regions of the injured tissue of the hMSC-treated femoral condyles. Moreover, in the hMSC-TCP group, the osteoclastic degradation of the scaffold and its replacement with mature bone was abundant in all regions of the defect, which was almost completely filled with mature bone tissue. These features were very rarely observed in the TCP-treated femoral condyles.
As already indicated, these qualitative histological observations were scored according to the parameters included in Table
Score of the subjects included in the study according to the evaluated histological parameters included in Table
Subject number | TCP (left femur) | hMSC-TCP (right femur) |
---|---|---|
1 | 9 | 25 |
2 | 8 | 22 |
3 | 10 | 23 |
4 | 9 | 22 |
5 | 7 | 19 |
6 | 12 | 24 |
7 | 13 | 23 |
8 | 6 | 19 |
9 | 9 | 20 |
10 | 10 | 22 |
11 | 10 | 23 |
12 | 7 | 18 |
13 | 11 | 25 |
14 | 6 | 19 |
Mean ± S.E.M. | 9.07 ± 0.57 | 21.71 ± 0.62 |
Representation of score values (vertical axis) of the subjects included in the study (horizontal axis). Score values for bone regeneration of TCP group (left femur) were clearly lower than those of the hMSC-TCP group (right femur).
The main aim of the current work was to ascertain if human MSC displayed their immunoprivileged properties in a xenogeneic setting of bone defect. In addition, we planned to compare in this setting the therapeutic effect of hMSC combined with a TCP-based carrier in a well-established model for critical-size defect. Interestingly, we have observed both the absence of inflammatory reaction in the implant area and a significantly higher bone regeneration ability of the hMSC-TCP group compared to the TCP-only group. These results may support the potential role of this combination in a clinical trial using allogeneic cells.
The rabbit model we have used for critical-sized defect of bone healing has been extensively used [
The human body has an extensive capacity to regenerate bone tissue after trauma. However, large defects cannot be restored without intervention and often lead to nonunion. Due to the multiple limitations associated with the use of autografts or bank-stored bones for bone reconstruction, investigators have developed alternative solutions. Recent tissue engineering approaches have attempted to create new bone based on seed MSC onto calcium phosphate ceramic scaffolds. Hydroxyapatite- (HA-) based ceramics presented slow resorption rate producing bone ingrowth onto a porous surface rather than a true bone regeneration [
Cell-based therapies are already used in musculoskeletal pathologies, such as bone fracture, pseduoarthrosis, and osteochondral defects [
Besides, it must take into account that MSC possess strong immune regulatory properties that are present in cells from different animal species, although with variable and only partially clarified mechanisms. MSC may suppress immune reactions
It has been reported that BMSC may be immune-privileged cells that do not elicit immune responses due to an absence of immunologically relevant cell surface markers. In addition, BMSC have immunomodulatory function. For that reason, BMSC theoretically can make them impervious to immunorejection following xenogeneic transplantation. Previous studies have reported opposite results ranging from no survival to differentiation into destination cells [
There are many works where xenogeneic MSC was transplanted into immunosuppressed animal models [
To our knowledge, this is the first demonstration of the survival of transplanted xenogeneic hMSC in a femur condyle defect and the bone formation without immune suppression. We observed that hMSC in combination with TCP granules successfully promotes bone formation in a critical-sized bone defect and the bone regeneration capacity was greater in comparison with TCP granules alone, where less bone regeneration process occurred. In the hMSC-TCP group, although bone regeneration of the defect was not complete, the presence of viable hMSC capable of osteogenesis was evident at 12 weeks in an immunocompetent rabbit model. Consistent with previous studies, hMSC combination with a scaffold resulted in significant bone formation when compared with scaffold only [
The results of the current study should be adequately interpreted taking into account some of the limitations of our work that include the small number of animals used and that the animals were analyzed histologically in a single time-point (after 12 weeks of implant).
In summary, our findings indicated that xenogeneic transplantation of hMSC using a calcium phosphate osteoconductive material promotes almost a complete regeneration of critical-size bone defect in an immunocompetent rabbit model. TCP granules can support proliferation and viability of hMSC. The incorporation of hMSC to TCP improves its osteointegration and bone regeneration. These results support the use of this combination in a nonautologous setting that should be explored in clinical trials.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflict of interest.