Bone Marrow Aspirate Concentrate versus Platelet ...

Original Article

Bone Marrow Aspirate Concentrate versus Platelet Rich Plasma to Enhance Osseous Integration Potential for Osteochondral Allografts Charles A. Baumann, BS1

1 Department of Orthopaedic Surgery/Thompson Laboratory for

Regenerative Orthopaedics, Missouri Orthopaedic Institute, University of Missouri, Columbia, Missouri J Knee Surg

Abstract

Keywords

► osteochondral allograft ► bone marrow aspirate concentrate ► platelet rich plasma ► orthobiologic

received February 20, 2017 accepted after revision May 3, 2017

James P. Stannard, MD1

James L. Cook, DVM, PhD1

Address for correspondence Aaron M. Stoker, MS, PhD, Department of Orthopaedic Surgery/Thompson Laboratory for Regenerative Orthopaedics, Missouri Orthopaedic Institute, University of Missouri, 1100 Virginia Ave., Columbia, MO 65212 (e-mail: [email protected]).

Fresh osteochondral allograft (OCA) transplantation is an attractive treatment option for symptomatic articular cartilage lesions in young, healthy patients. Since a lack of OCA bone integration can be a cause of treatment failure, methods for speeding and enhancing OCA bone integration to mitigate this potential complication are highly desirable. This study sought to determine and compare the potential of bone marrow aspirate concentrate (BMC) and leukoreduced platelet rich plasma (PRP) to repopulate the osseous portion of an OCA with cells and deliver osteogenic proteins. It was hypothesized that BMC would have significantly higher colony forming units (CFUs)/mL and seed the osseous portion of OCA with more cells than PRP. Finally, we hypothesized that the media of BMC and PRP treated OCAs would have significantly higher concentrations of osteogenic proteins compared with negative control OCAs. Cylindrical OCAs (n ¼ 36) created from tissue stored for 21 days were treated with BMC (n ¼ 12) or PRP (n ¼ 12) obtained for 6 dogs, or left untreated as a negative control (n ¼ 12). After treatment, OCAs were cultured for 7 or 14 days. Media were collected for analysis of osteogenic biomarker concentration. Samples of each BMC and PRP were tested for CFU concentration. On day 7 or 14, the grafts were assessed for cell surface adhesion and penetration using fluorescent microscopy. Significant differences in CFU and media biomarker concentration the between groups were determined using oneway analysis of variance (ANOVA) and Tukey’s post-hoc test with the significance set at p < 0.05. Only OCAs saturated with BMC had viable cells detectable on the osseous portion of the allografts at day 7 and 14 of culture. BMC samples had a significantly higher (p ¼ 0.029) CFU/mL compared with PRP samples. At day 3 and/or 7 of culture, the concentration of several osteogenic proteins was significantly higher in both BMC and PRP samples. Autogenous BMC can be used to deliver both a cell population and osteogenic proteins that may improve healing of the osseous portion of the OCA clinically.

Copyright © by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.

DOI https://doi.org/ 10.1055/s-0037-1603800. ISSN 1538-8506.

Downloaded by: University of Missouri-Columbia. Copyrighted material.

Aaron M. Stoker, MS, PhD1

Stoker et al.

Articular cartilage lesions are identified in up to 66% of patients undergoing knee arthroscopy with approximately 20% of these lesions being considered high grade.1,2 Osteochondral allograft (OCA) transplantation is an effective treatment option for large chondral and osteochondral defects in the knee, hip, ankle, and shoulder with a 10-year survivorship success rate as high as 85% in patients. One of the causes for OCA failure is incomplete integration of donor allograft bone into the recipient bone. Because the OCA bone is devoid of viable cells and blood supply at the time of implantation and is allogeneic, its integration into the recipient bone is dependent upon cellular repopulation and neovascularization from the patient via creeping substitution, which is a slow process.3,4 Enhancing this process using orthobiologics such as autogenous bone marrow aspirate concentrate (BMC) or platelet-rich plasma (PRP) could minimize failures and improve graft survivorship and patient outcomes. PRP can be produced by various methods and is a source of platelet-derived growth factors (PDGFs) with angiogenic and osteoinductive potential5,6 and cells.7 It has been evaluated in numerous in vivo animal model studies for its potential to enhance bone healing7–11 and OAC and autograft healing.12,13 The results from these studies have been variable with some findings improving bone healing with PRP treatment and others with no significant benefits for PRP treatment with respect to bone healing. One factor that may contribute to the variability in the outcomes of these studies is the absence of a standard method for the production of PRP for clinical use, resulting in PRP preparations with variable concentrations of platelets, leukocytes, and proteins.14–16 One in vitro study found that leukocyte-poor PRP had lower inflammatory cytokine concentrations, higher cell proliferation, viability, and migration compared with leukocyte-rich PRP; this indicates that leukocyte-poor PRP may be more appropriate for enhancing bone healing.14 Bone marrow aspirate (BMA) contains pluripotent mesenchymal stem cells, which have the ability to respond to autocrine and paracrine stimulation at a wound site to expedite healing.17–21 However, the concentration of these stem cells in the BMA is low, and therefore, requires enrichment to obtain clinically relevant improvements in wound healing.17–19,22 BMC can be produced at the bedside during a surgical procedure by concentrating a BMA obtained from a patient through a centrifugation or filtration protocol.18,23–25 The concentration process increases the nucleated cell concentration of the BMC by 2 to 40 times over that of the original bone marrow aspirate.18,25,26 The stem cells in BMC have the potential to become osteoblasts18,27 and can respond to local stimuli to produce new bone and recruit additional cells to stimulate the healing, integration, and remodeling processes. Additionally, BMC has been reported to contain many of the same growth factors as PRP including PDGF, transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF).21,22 Osteoinductive proteins create an anabolic anti-inflammatory environment that may accelerate and promote the process of bone healing7,28,29 and OCA bone integration.30,31 Because both BMC and leukoreduced PRP are considered autologous orthobiologics that can be used clinically to The Journal of Knee Surgery

enhance bone healing,7 this study was designed to compare BMC and PRP for their potential in enhancing osseous integration of OCAs clinically. This evaluation was based on the osteoprogenitor cell retention in the bony portion of the OCA and relevant protein concentration in the culture media. The study hypothesis was that BMC would provide superior osseous integration potential for OCAs when compared with PRP and saline-based on viable osteoprogenitor cell repopulation of grafts and osteoinductive protein production in a canine ex vivo model.

Materials and Methods Collection of BMC and PRP All procedures were performed with institutional care and committee approval. From each research dog (n ¼ 6), both a BMC and PRP sample were collected for this study. BMC and PRP collections were performed under sedation and aseptic conditions. Canine BMC and PRP were prepared using a commercially available system (Angel System, Arthrex, Naples, FL), which was validated in previous studies to produce BMC and leukoreduced PRP using the following methods. For BMC, 50 mL of BMA with 10 mL of anticoagulant citrate dextrose, solution A (ACD-A) was collected from the proximal humerus of adult purpose-bred research dogs (n ¼ 6) using a Jamshidi needle and 30 mL syringes, and processed at the 7% setting on the Angel machine. For PRP, 40 mL of whole blood was collected with 5 mL of ACD-A from the jugular vein of adult purposebred research dogs (n ¼ 6), and processed using the 2% setting on the Angel machine. Immediately after processing using the Angel machine, BMC and PRP were used to completely saturate the osseous portion of OCAs, as described below.

Tissue Harvest and Culture Distal femurs were harvested from adult purpose-bred research dogs (n ¼ 3) immediately after euthanasia for reasons unrelated to this study and stored for 21 days using the Missouri Osteochondral Allograft Preservation System (MOPS) protocol. On day 21 of preservation, cylindrical OCAs (8 mm diameter 8 mm depth) were created (n ¼ 36; 12/dog) and randomly assigned to one of the following treatment groups: 1. Standard of care (SOC)—the bone portion of each OCA was thoroughly irrigated with 10 mL of saline as per clinical SOC. 2. BMC—the bone portion of each OCA was thoroughly irrigated with 10 mL of saline, dried with a sterile sponge, and saturated with 0.5 mL of BMC. 3. PRP—the bone portion of each OCA was thoroughly irrigated with 10 mL of saline, dried with a sterile sponge, and saturated with 0.5 mL of PRP. The BMC and PRP samples collected from each dog were used to treat two OCA plugs, one for each time point of culture (day 7 or 14). Therefore, at each time point, there was an OCA plug treated with a BMC or PRP sample from each dog. The OCAs were placed into 6-well culture plates containing 8 mL of supplemented Dulbecco’s Modified Eagles Medium (DMEM with 10% fetal bovine serum [FBS], sodium


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pyruvate, penicillin, streptomycin, and amphotericin B) and cultured for 7 or 14 days (n ¼ 6/group/day). The media were changed on days 3, 7, 11, and 14 and collected on days 3, 7, and 14 for biomarker analysis. On days 7 and 14, OCAs (n ¼ 6/group/time point) were evaluated for cell colonization and infiltration, as described below.

Viable Cell Colonization

Media Analysis The OCA culture media on days 3, 7, and 14 were assessed for alkaline phosphatase (ALP), Dickkopf-1 (DKK-1), osteoprotegerin (OPG), osteopontin (OPN), adrenocorticotropic hormone (ACTH), bone morphogenic protein-2 (BMP-2), and bone morphogenic protein-7 (BMP-7) using canine specific commercially available assays (MyBioSource, Inc., San Diego, CA) according to manufacturers’ protocol.

Colony Forming Unit (CFU) Analysis For CFU analysis, the BMC (n ¼ 6) and PRP (n ¼ 6) remaining for each sample after seeding of OCAs were pelleted at 1,000 g for 10 minutes. The supernatant was removed, the pellets were washed once in 10 mL of DMEM, centrifuged again at 1,000 g for 10 minutes, and the cell pellet resuspended in 10 mL of supplemented DMEM. Then, 0.125, 0.25, 0.5, and 1.0 mL aliquots of the resuspended pellets were plated onto 75 cm2 tissue culture flasks (n ¼ 3/sample/volume) and cultured in a final volume of 20 mL of supplemented DMEM, as described above. The range of volumes was used to ensure that individual colonies could be observed for each sample set. The samples were cultured for 7 days, stained with 1% crystal violet for 10 minutes, rinsed twice with PBS, and the number of CFUs for each sample was determined by manually counting the number of individually stained colonies on each flask. The number of CFUs per flask counted was standardized to CFUs/ mL, and the average of all flasks was used for the CFU value for that sample. Data are reported as number of CFUs/mL for each sample.

Data Analysis Media biomarker and CFU data were compared for statistically significant differences among groups using one-way ANOVA and Tukey’s post-hoc test with significance set at p 0.05.

Fig. 1 Mean standard error (SE) colony forming units (CFUs)/mL) for bone marrow concentrate (BMC) and platelet rich plasma (PRP). p < 0.05, significantly higher than PRP.

Results CFU Analysis All BMC samples had detectable CFUs while only one PRP sample had detectable CFUs. The number of CFUs/mL was significantly (p ¼ 0.029) higher in the BMC samples compared with the PRP samples, indicating that BMC had a higher potential for cell repopulation of the osseous portion of the OCA graft compared with PRP (►Fig. 1).

Viable Cell Colonization The osseous portion of all OCAs was subjectively assessed for the presence and distribution of viable cells at day 7 and 14 of culture. At both time points, only OCAs saturated with BMC had viable cells detectable on the surface of the osseous portion of the cultured OCAs. Further, viable cells had penetrated into the osseous portion of all BMC treated OCAs and were observable in the deep area of the osseous portion at days 7 and 14 of culture. Viable cells were not observed in any part of the osseous portion of PRP or SOC OCAs at either time point (►Fig. 2A-L).

Media Analysis Concentrations of BMP-2 were significantly higher in the BMC group at days 3 (p < 0.001) and 7 (p ¼ 0.017) and the PRP group at day 3 (p ¼ 0.009) compared with the SOC group. The concentration of DKK-1 was significantly (p ¼ 0.038) higher in the BMC group compared with the SOC group at day 3. Concentrations of OPG were significantly higher in the BMC group at days 3 (p < 0.001) and 7 (p 0.002) and the PRP group at day 3 (p < 0.001) compared with the SOC group. Further, the concentration of OPG in the BMC group was significantly higher than the PRP group at day 7 (p 0.004) of culture. The concentration of ALP was significantly lower in the PRP group compared with the SOC group at day 3 (p ¼ 0.03). There were detectable concentrations of ACTH in all groups, but there was not a significant difference among groups at any of the time points tested. Concentrations of BMP7 and OPN were below the detectable limits of the assay for all groups at both time points (►Fig. 3A-E). The Journal of Knee Surgery


On days 7 and 14, the osseous portion of each OCA was analyzed for viable cell attachment and infiltration using the fluorescent live stain, calcein acetoxymethyl (AM). Tissues were incubated in phosphate buffered saline (PBS) containing calcein AM (10 µM) for 30 minutes at 37°C. After staining, tissues were washed in PBS for 5 minutes at room temperature to remove excess stain. The outer surface of the osseous portion of OCA was then subjectively assessed using an Olympus BX-51 fluorescent microscope. Images were taken at magnification 4 using an F-view CCD cooled fluorescent camera and the program MicroSuite (Olympus Corp, Shinjuku, Tokyo, Japan). After the graft surface was evaluated, the graft was cut into half to assess the penetration of viable cells into the osseous portion of the OCA during culture. Tissues were stained and assessed, as described above.


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Discussion Over the last four decades, OCA transplantation has gained traction as a treatment modality for large cartilage defects of the knee, as well as the ankle, hip, and shoulder. Previous reports validate the safety, efficacy, and survivorship of this treatment option.32–34 While numerous studies have focused on the importance of chondrocyte viability,35–38 there has been relatively little focus on the osseous integration of these grafts following transplantation. Because the OCA bone is devoid of viable cells and blood supply at the time of implantation and is allogeneic, its integration into recipient bone is dependent upon cellular repopulation, revascularization, and matrix replacement and remodeling through the plodding process of creeping substitution.3,4 Unfortunately, the prolonged timeline for completion of this complicated process elevates the risk for graft failure when integration cannot keep pace with the biomechanical and biologic demands of the graft. Impending or definitive failure of allograft bone integration is diagnosed based on clinical symptoms in conjunction with radiographic findings of radiolucency at the graft-host border, graft subsidence,

contour irregularities, and/or loss of normal trabecular architecture of the grafted condyle, including subchondral cysts and/ or moderate to severe sclerosis.39–41 As such, techniques aimed at enhancing and accelerating the process of OCA bone integration are vital to improving patient outcomes. It is theorized that delivery of an autologous cell population with osteogenic potential would enhance the integration of the bone portion of the OCA into the recipient tissue and potentially decrease OCA transplantation failures resulting from poor integration of allograft bone. In agreement with previous data,26 BMC used in the present study consistently contained osteoprogenitor cells that were capable of repopulating the osseous portion of OCAs. Viable cell repopulation of OCAs did not occur for any PRP- or SOC (saline)-treated grafts, indicating that only BMC provided the potential for enhancing OCA bone integration by seeding OCAs with autologous osteoprogenitor cells. The other mechanism by which orthobiologics may enhance OCA integration is through chemotactic, neovascularization, and osteoinductive growth factors, cytokines, and other proteins contained in PRP and BMC. While the concentrations of

Fig. 3 Mean standard error (SE) media concentration on days 3, 7, and 14 of culture: (A) bone morphogenic protein (BMP)-2. (B) dickkopf (DKK)-1. (C) adrenocorticotropic hormone (ACTH). (D) osteoprotegerin (OPG). (E) alkaline phosphatase (ALP). p < 0.05, significantly higher than standard of care (SOC) at time point; †p < 0.05, significantly lower than bone marrow concentrate (BMC) at time point; and °significantly lower than SOC at time point. The Journal of Knee Surgery


Fig. 2 Representative viable cell colonization pictures (magnification 4) at day 7 (A-F) and 14 (G-L) showing viable cell content across the surface (top) and in the center of the osseous portion of the graft (bottom). (A) bone marrow concentrate (BMC)-7, (B) BMC-7-center, (C) platelet rich plasma (PRP)-7, (D) PRP-7-center, (E) standard of care (SOC)-7, (F) SOC-7-center, (G) BMC-14, (H) BMC-14-center, (I) PRP-14, (J) PRP-14-center, (K) SOC-14, and (L) SOC-14-center. Arrows indicate viable staining cells on the interior of the osseous portion of the graft.


for BMC to enhance osteochondral allograft bone integration to minimize failures and improve graft survivorship and patient outcomes are warranted.

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PDGF, TGF-β, and VEGF have been evaluated in BMC and PRP, those of biological factors associated with bone integration and remodeling have not been reported to the authors’ knowledge. Therefore, the present study examined concentrations of BMP2, OPG, ACTH, BMP-7, OPN, DKK-1, and ALP activity eluted from OCAs treated with PRP, BMC, or saline after 3, 7, and 14 days in culture. BMP-2 initiates bone formation and healing, induces synthesis of other BMPs,42 induces osteoblast differentiation of bone marrow stromal cells,43 and may increase angiogenesis by increasing production of VEGF.44 Additionally, it has been shown to increase ALP activity and bone mineralization by osteogenic cells.45 In the present study, BMC and PRP treated OCAs eluted significantly higher levels of BMP-2 compared with saline-treated controls with the BMC group eluting the highest concentrations, which were also sustained, compared with the PRP group. OPG is critical to early bone healing and remodeling and inhibits bone resorption and osteoclastogenesis.42,46–48 In the present study, BMC and PRP treated OCAs eluted significantly higher levels of OPG compared with saline-treated controls with the BMC group maintaining significantly higher levels for the entire study period. ACTH supports osteoblast activity, increases VEGF production,49 and helps maintain bone mass.50,51 In the present study, ACTH was detectable in all groups, but no significant differences were noted among treatments. ALP plays important roles in osteoblast differentiation, osteogenesis, and calcification.52–57 While all OCA treatments were associated with ALP elution, the lack of significant differences among treatment types suggests that the majority of ALP came from the graft itself and neither BMC nor PRP enhanced this biologic factor. DKK-1 inhibits bone healing58–60 by blocking binding of wingless-int (WNT)/β-catenin to WNT coreceptors, lipoprotein-related proteins 5 and 6.59,61 In this study, BMC treated OCAs eluted significantly higher levels of DKK-1 compared with saline-treated controls at day 3. However, media concentrations of DKK-1 were still below 1 ng/mL, which is far below the mean serum concentrations reported for normal individuals of 10 to 31 ng/mL.62,63 Therefore, it is not clear if the media concentrations of DKK-1 observed in this study are clinically relevant. BMP-7 and OPN were not eluted at detectable levels from any treatment group in this study. The authors recognize the limitations to this study. This study assessed canine OCAs ex vivo such that clinical effects of the differences noted cannot be determined from these data. In addition, characterization of the composition of BMC and PRP were based on previously reported data. However, with these limitations in mind, the data from the present study allow us to accept the hypothesis that BMC provides superior osseous integration potential for OCAs when compared with PRP and SOC saline treatment. Treatment with BMC in a clinically relevant manner resulted in repopulation of the osseous portion of the graft with viable osteoprogenitor cells. Further, BMC treatment was associated with a robust and sustained release of biologically active proteins known to enhance bone integration and remodeling. Based on these findings, clinical studies assessing the capabilities

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