Response of the Bone Marrow Stromal Cells to Osteogenic Drugs in Patients Undergoing Primary Total Hip Arthroplasty

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Response of the Bone Marrow Stromal Cells to Osteogenic Drugs in Patients Undergoing Primary Total Hip Arthroplasty

Corresponding author: Weiping Ren MD Ph.D, Department of Biomedical Engineering, Wayne State University College of Engineering, 6135 Woodward Avenue, Detroit, MI 48202, Tel: (313)577-1383 ;Fax:(313)577-8333 ; Email: as7606@wayne.edu Received: 05-11-2017

Abstract

Implant osseointegration relies on the interplay of implant surface and the surrounding bone cells. The purpose of this study was to investigate the in vitro response of the bone marrow stromal cells (BMSCs) to osteogenic drugs in patients undergoing pri- mary total hip arthroplasty (THA). Bone marrow aspirates were obtained from the discarded femoral head in 13 THA patients. The number of STRO-1 and bone morphogenetic protein receptor (BMPR)-expressing cells were measured by flow cytometry. Real-time PCR was used to measure expression of osteogenic relevant genes of BMSCs. Osteogenic potential was evaluated by alkaline phosphatase (ALP) activity. The response of BMSCs to osteogenic drugs (BMP-2, dexamethasone and etidronate) was further evaluated. There was a strong association between the gene levels of BMPR1a, ALP, RunX-2 and MSX2 with the percent of STRO-1+ cells. BMP-2 (100 ng/ml) stimulated the BMPR-pSMAD1/5 signaling pathway. Both etidronate (250 ng/ml) and dexamethasone (10 nM) treatments raised ALP activity in 33% and 70% of BMSC samples, respectively. This was independent of the activation of pSMAD signaling. A better understanding of the individual variations of BMSCs in response to osteogenic drugs is important for the THA patient management and the strategic plan of implant surface drug delivery.

Keywords: Bone Marrow Mesenchymal Stromal Cells; Total Hip Arthroplasty; Osseointegration; Bone Morphogenetic Protein; Flow Cytometry; Alkaline Phosphatase (ALP) Activity 

Introduction

More than 500,000 total hip arthroplasty (THA) operations are performed in the USA annually[1]. Cementless THA is rap- idly being accepted as the surgery for arthritic diseases of the hip joint [2]. Implant stability and longevity require efficient osseointegration, which is a process requiring the recruit- ment of bone marrow mesenchymal stromal cells (BMSCs) to the prosthesis surface, resulting in the direct anchorage of an implant by bone formation at the bone-implant surface. The commitment of BMSCs to an osteoblastic differentiation path- way is under the control of both systemic and local growth factors, such as bone morphogenetic protein 2 (BMP-2). Cur- rent research focuses on the interplay between implant [3] and periprosthetic environment [4]. A favorable bone marrow

environment should have a sufficient number of osteoprogen- itor cells able to differentiate into functional osteoblasts in response to growth factors [5]. A better understanding of the genetics and responses of BMSCs of THA patients to osteogen- ic drugs is important but not well defined [5,6].

STRO-1 is a cell surface antigen expressed by BMSCs [7]. The bone marrow STRO-1+ cells are capable of differentiating into multiple mesenchymal lineages including adipocytes, osteo- blasts, and chondrocytes [7]. The variation of STRO-1+ cells can be observed between human subjects [8]. We have identi- fied that there are no differences in Stro-1+ cells between male and female patients, though there are slight increases in the number of Stro-1+ cells in younger patients (<70 years old) than those of older patients (≥70 years old). Therefore, our

data suggest that the age and gender of THA patients have little impact on the numbers of Stro-1+ BMSCs (4).

BMPs are a group of growth factors known for their ability to induce bone formation [9]. BMPs activate target cells by bind- ing to type-Ia, -Ib and -II BMP receptors (BMPRs) [9]. BMPR1a is necessary for the extracellular matrix deposition by osteo- blasts [10]. Type II receptor (BMPR2) binds BMPs and then initiates intracellular signaling, including the recruitment and phosphorylation of cytoplasmic proteins, especially the ‘sig- naling mothers against decapentaplegic’ (SMAD) 1, 5 and 8 [11]. Osyczka et al.[12] found that human BMSCs exhibited a lower in vitro osteogenic response to BMP-2 compared to ro- dents. However, it is still unclear whether disparate BMSC re- sponses to BMP also apply to other osteogenic molecules, such as bisphosphonate [13] and dexamethasone [14].

A detailed “bone marrow osteogenic potential” analysis of THA patients might be helpful for surgeons to predict the perfor- mance of implants after surgery. An inferior bone marrow en- vironment (such as insufficient BMSCs, lower response of BM- SCs to osteogenic molecules, etc.) may represent a risk factor for the dysfunctional osseointegration and earlier implant fail- ure. The purpose of this study was to investigate the response of BMSCs collected during primary THA procedures to differ- ent osteogenic drugs.

Materials and Methods

Patients

A total of 13 patients scheduled to undergo cementless THA were consecutively recruited for study (8 men and 5 women, 50-84 years old, body mass index (BMI) ranged from 21.5 to

40) in the Department of Orthopaedic Surgery at Providence Hospital. The Clinical Investigation Plan and the Informed Consent Form were approved by the Ethics Committee of the hospital. Patients were entered into the study after reading and signing an informed consent form. Criteria for exclusion were rheumatoid arthritis, autoimmune disease, or symptoms or signs of infection or inflammation. Also excluded were pa- tients taking nonsteroidal anti-inflammatory drugs within one month before examination.

Human BMSCs

Bone marrow aspirates were obtained from the metaphyseal region of the discarded femoral head during THA. After filtra- tion of the bone marrow aspirates through a 45 m-pore size cell strainer (Falcon, BD Biosciences, Bedford, MA), the bone marrow cells were collected and diluted 1:3 with Hanks’ Bal- anced Salt Solution and centrifuged at 200xg for 10 minutes. Supernatants were stored at -80ºC and cell pellets containing both red and white blood cells were plated in 75 mm flasks with alpha-modified Eagle’s medium (MEM) containing 20% fetal bovine serum (FBS, Thermo Scientific, Logan, UT), 2 mM

glutamine, penicillin (100 U/ml), streptomycin sulfate (100 ug/ml) and incubated at 37ºC in a humidified atmosphere of 5% CO2. Aspirates were generally plated at a higher density (up to 20 X 107/ml) because of the dilution of marrow with peripheral blood. After 2 days of culture, red blood cells were washed off the attached cells and media was replaced with media containing 10% FBS. Once the cultures reached 50-70% confluence they were passaged. When these passage 1 (P1) cells reached 70% confluence, they were harvested using Tr- pLE Express (Gibco, Grand Island, NY) and frozen or used to determine the percent of STRO-1+ cells in the culture.

Flow cytometry

Detection of cell surface markers (STRO-1, BMPR1a and BMPR2) was performed using an Accuri C6 flow cytometer (Accuri, Ann Arbor, MI) equipped with CFlow software. The BMSC suspension was incubated with primary antibodies for 1 hour at 4°C followed by thirty minute incubation with secondary antibodies. Mouse IgG anti-human STRO-1 (1 ug #MAB1038, R&D, Minneapolis, MN) was used with allophyco- cyanin conjugated rat anti-mouse (#550676, BD Biosciences). For goat anti-human BMPR1A (1 ug #AF3460, R&D) and for goat anti-human BMPRII (1ug #AF811, R&D) the secondary antibody was phycoerythrin conjugated donkey anti-goat IgG (#F010, R&D). At least 10,000 cells were analyzed per sam- ple. Normal species specific IgG1 was used as a negative con- trol. After incubation, cells were washed and resuspended in 500 l of wash buffer and measured by flow cytometry. For the measurement of intracellular expression of pSMAD 1/5/8, cells were collected after 30 minutes of drug treatment and fixed with 2% formaldehyde for 10 minutes at 37°C. The fixed cells were then permeabilized with ice-cold 100% methanol. The permeabilized BMSCs were incubated with primary an- ti-pSMAD 1/5/8 antibody (14 ng, Cell Signaling #9516S, rab- bit anti-human IgG) or isotype control normal rabbit IgG for 1 hour at 4oC. Unbound antibodies were removed by washing using wash buffer. Secondary antibody Alexa Fluor 488 con- jugated-goat anti-rabbit (Cell Signaling #4412) were applied for 30 minutes. After incubation, cells were washed and re- suspended in 500 ul of wash buffer and measured by flow cy- tometry. All data were analyzed using CFlow software (Accuri). The average fluorescence intensity (AFI) of the cell population was calculated for each antigen after subtraction of the AFI of the negative control samples from the AFI of the test samples (cells treated with the specific antibody).

Real time polymerase chain reaction (PCR) analysis

RNA was isolated from the first passage cells and used for Real Time PCR measurement of gene expression (StepO- nePlus System, Applied Bio systems, and Forest City, CA). Primers (Invitrogen, Carlsbad, CA) for Alkaline Phospha- tase (ALP): 5’-ACCATTCCCACGTCT TCACATTG-3’ (forward) and 5’-AGACATTCTCTCGTTCACCGCC-3’ (reverse), BMP-2: 5’-GAGTTGCG-GCTGCTCAGCATGTT-3’ (forward) and 5’-ACAT-

GTCTCTT GGAGACACCT-3’ (reverse), BMPR1a: 5’-CATAACTA- ATGGACATTGCT-3’ (forward) and 5’-GCAGCTGGAGAAGA

TGATCATAGC-3’ (reverse), Msh Homeobox-2 (MSX-2): 5’-GC- CATTTTCAGCTTTTCCAG-3’ (forward) and 5’-CCCTGAGGAAA-

CACAAGACC-3’ (reverse), Runt-related transcription factor 2 (Runx2): 5’-AGATGATGACACTGCCACCTCTG-3’ (forward) and

5’-GGGATGA- AATGCTTGGGAACTGC-3’ (reverse), Glyceralde- hyde 3-phosphate dehydrogenase (GAPDH): 5’-GAGCCACATC- GCTCAGACAC-3’ (forward) and 5’-CATGTAGTTGAGGTCAAT-

GAA GG-3’ (reverse) used Sybr Green Master Mix. Taqman assays were used for Sclerostin (SOST) (HS00228830m1), BMPR1b (HS00176144m1), and GAPDH (HS99999905m1)

(Applied Biosystems).

To standardize the target gene level with respect to varia- tions in RNA and cDNA, the housekeeping gene GADPH was used as an internal control. To determine the relative level of gene expression, the comparative threshold cycle (CT) meth- od with arithmetic formulae were used. Subtracting the CT of the housekeeping gene from the CT of target gene yielded the ΔCT for each gene. Choosing one patient sample as the control norm, all other samples were compared to determine if they had more, less or similar expression of the same gene. This cal- culation yielded a ΔΔCT which was then converted to 2(-ΔΔCT) for the exponential amplification of gene expression.

Analysis of osteogenic potential of P1 BMSCs

Osteogenic potential was measured by cellular ALP activity (BioVision Kit #K412-500) after culturing BMSCs in osteogenic medium (α-MEM/10% FBS supplemented with 100 nM dexa- methasone (DX, D2915, Sigma), 10 mM glycerol 2-phosphate and 284 μM L-ascorbic acid) for 14 days. ALP activity in the cell lysate was measured spectrometrically at 405 nm (Syner- gyHT by BIO-TEK, Winooski, VT) utilizing the conversion of a colorless p-nitrophenyl phosphate to a colored p-nitrophenol (Sigma, St. Louis, MO). ALP levels were normalized to protein concentration (DC Protein Assay, BIO-RAD) allowing ALP ac- tivity to be expressed as μM pNPP/mg protein/min.

Drug treatment

For this study, frozen P2 BMSCs were culture-expanded in basal MEM/10% FBS medium. P3 cells, 50% confluent, were serum starved (1% FBS) overnight and then exposed to either 100 ng/ml BMP-2 (Cell Signaling, 4697), 250 ng/ml etidronate (P5248, Sigma) or 10 nM DX (Sigma) in media supplemented with 10 mM β-glycerolphosphate and 284 mM L-ascorbic acid. Cells collected after 30 minutes or 24 hours were used for flow cytometry. Cells collected after 21 days of culture were used for ALP activity and Alizarin Red staining (#ECM815, Osteo- genesis Assay Kit, Millipore). BMSCs without drug treatment were included as controls. Culture media and drugs were replaced every three days until the predetermined time of harvest.

Statistical Analysis

Data were expressed as mean ± SE. Group mean values were compared using one-way analysis of variance with Newman– Keuls test if a normal model could be set up; if not, the Mann– Whitney U test was used. The Spearman’s test was used for correlation analysis, with P values less than 0.05 considered to be statistically significant.

Results

ALP activity of BMSCs

We measured ALP activity following 21 days of treatment of cultured cells with 10 mM β-glycerolphosphate and 284 mM L-ascorbic acid. The mean activity (+ SE, n=12) was 54.8 ± 11.9 U/mg protein. There was considerable difference in ALP ac- tivities among cultures of patient samples (Figure 1). There was no significant relationship between ALP activity and the percent of BMSCs positive for STRO-1 (R=0.528, P=0.077). In addition no relationship between ALP activity and the age, sex as well as BMI of the subjects was observed (data not shown).

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μM pNPP/mg protein/minute

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Figure 1. ALP activity of BMSCs from bone marrow aspirates cultured in osteogenic media for 21 days (n=12).

Yield of STRO-1+ Cells from BMSCs

Cells cultured from thirteen different patient samples of bone marrow aspirates were analyzed for STRO-1+ cells by flow cytometry after P1. The mean percent yield (± SE) of STRO-1+ cells was 9.4% ± 16.5%, indicating a great deal of variability among patients (Figure 2). There was no significant difference in the percent of STRO-1+ cells between men (11.9% ± 7.2%, n=8) and women (5.4% ± 3.5%, n=5). Similarly, there was no significant correlation between the % of STRO-1+ cells and a patient’s age or BMI.

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Figure 2. The percent of passage one cultured BMSC stained with an- tibody to STRO-1 and measured in Accuri C6 flow cytometer shows a high degree of variability between these thirteen total hip replace- ment patients.

Association of osteogenesis-relevant gene expression with ALP activity and STRO-1+ cells

To investigate the osteogenic potential of BMSCs, we measured a panel of genes, including Runx2, MSX2, BMPR-1a, BMPR-1b, BMP-2, ALP and SOST by real time PCR using mRNA isolated

signal transduction initiated by binding of BMP to BMPR. In order to determine whether the pSMAD signaling pathway was activated in response to osteogenic drug treatment, we harvested cells for flow cytometry 30 minutes after admin- istration of BMP-2, etidronate or DX. Not surprisingly, BMP-2 gave the strongest response with measurement of intracellular pSMAD up to 4-fold higher than control treatments of ascorbic acid and β-glycerol phosphate (Figure 4a). The average stimu- lation was 275%. Etidronate and DX did not significantly stim- ulate pSMAD intracellular expression. The percent of different patient cell cultures responding to drugs is given in Table 1.

from P1 BMSCs. Consistent with the data for ALP activity and STRO-1+ expression, the BMSC samples exhibited a wide range of gene expression. However, a significant positive correlation was found between the expression of four genes (ALP, RunX2, BMPR1a and MSX-2) and the percent of STRO-1+ cells detected by flow cytometry (Figure 3). In addition, an association was observed between gene expression of ALP and ALP enzyme ac- tivity. There was no association between ALP enzyme activity and other osteogenesis-relevant genes measured.

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  1. pSMAD

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BMPR1A

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R=0.665 P=0.018

R=0.962 P=0.0001

ALP gene expression

Runx2 gene expression

Percent change from control

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BMP2 Etidronate DX BMP2 Etidronate DX 400

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R=0.701 P=0.011

R=0.634 P=0.027

BMPR1A gene expression

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Figure 4. Treatment with the osteogenic drug, BMP2 increases the pSMAD signaling relative to control and to other treatments in BMSC cultured for 30 minutes (a). BMP2 as well as the other drugs, etidro- nate and DX, have a more variable stimulating effect on the cell surface receptor, BMPR1A after a 2 day exposure to the drugs (b). Dexameth- asone (DX) treatment for 21 days stimulates alkaline phosphatase ac- tivity as well as the subsequent calcium phosphate deposits revealed by Alizarin Red staining (c,d).

BMPR response to osteogenic drugs

Percent STRO-1 labeled cells Percent STRO-1 labeled cells

Figure 3. Correlation analysis between osteogenic gene expression (2 (–ΔΔCT) for ALP, Runx2, BMPR1A and MSX2) and the percent of STRO-1+ cells in BMSC cultures from 12 THA patients The correlation coefficient (R) analysis was done by SPSS bivariate correlation (Pear- son) analysis.

Activation of pSMAD signaling in response to osteogenic drugs

SMADs regulate transcription of BMP-responsive genes. Phos- phorylation of SMAD1/5/8 is a major, early activation step in

Since binding of BMP to its cell surface receptor is required for initiation of the pSMAD signaling cascade, we investigat- ed changes in this receptor following two days of osteogenic drug treatment. Detection of the BMPR2 cells surface antigen proved difficult and the data did not provide any conclusive results indicative of a change in receptor level following two days of osteogenic drug stimulation (data not shown). An in- crease in BMPR1a expression on the cell surface after two days of treatment could be seen in only a small percentage of pa- tients in response to BMP-2, etidronate or DX (Table 1). The average magnitude of this change was 135% of control and it was extremely variable from culture to culture leading to very

large standard error OR DEVIATION? (Figure 4b).

pSMAD+ cells BMPR1a+ cells ALP activity
BMP 2 77% 20% 27%
Etidronate 9% 10% 33%
Dexamethasone 17% 30% 70%

Table 1. Response of BMSC cultures to osteogenic drugs. A positive response was defined as a value greater than 125% of control-treated cells. Of the twelve samples tested 77% responded to BMP2 with an increase in pSMAD signaling (275 + 39 percent increase over control, mean + SE, n=12) where as 70% responded to DX with an increased ALP activity (190 + 33 percent increase over control, mean + SE, n=10).

Figure 5. BMSC sample #5 cultured in osteogenic medium for 21 days. Control (a), etidronate (b), BMP2 (c), DX (d).

ALP activity and Alizarin Red staining of BMSCs in response to osteogenic drugs

To determine whether and how BMSCs samples are respon- sive in a functionally relevant manner to osteogenic drugs, P3 BMSCs were treated with BMP-2, etidronate or DX. Cells were collected after 21 days of culture. The ALP activity in BMSCs without drug treatment was used to normalize values for each patient culture. A positive drug response was considered to be an ALP activity greater than 125% of control ALP. As shown in Figure 4c, an increased ALP activity was observed in some

but not all P3 BMSCs samples with a response rate for BMP-2 (27%), etidronate (33%) and DX (70%), respectively (Table 1).

Alizarin red S staining has been used for decades to evaluate calcium-rich deposits by cells in culture[15]. Alizarin Red S analysis was used to assess the mineralization of drug-treated cells (Figure 5). As shown in Fig. 5d, adding DX significantly enhanced mineralized nodule formation. However, BMP-2 treatment only slightly increased mineralization as compared to controls (Figure. 5C), and etidronate treatment had no effect (Figure.5B).

Discussion

There are conflicting results regarding an association of THA success rate with patient age, sex, or body weight [16, 17]. This may, in part, be due to the influences of ethnic status, general disease background, physical activity level, [18] and surgical variables. A better understanding of a patient’s bone marrow microenvironment profiles is important but has not been well studied. In this study, we demonstrated that there are highly diversified BMSC profiles characterized by the percentage of STRO-1+ cells, ALP activity and osteogenic-relevant gene pro- files in this group of 13 THA patients.

STRO-1+ is a cell surface antigen expressed in human bone marrow cells. A STRO-1+ enriched subset of BMSCs is capa- ble of differentiating into multiple mesenchymal lineages in- cluding adipocytes, osteoblasts and chondrocytes [19]. The percent of BMSC STRO-1+ cells has been shown to vary with age, sex [8, 20], and osteoarthritis background [21]. It is still unclear whether the variation of the BMSC STRO-1+ cells rep- resents a biomarker for BMSC osteogenic potential.

Osteoblast differentiation is regulated by a panel of transcrip- tion factors such as Runx2 and MSX2 [22]. Runx2 commenc- es the expression of key osteoblast proteins such as ALP and osteocalcin, and is expressed in bone cells throughout the osteogenic lineage [22]. MSX2 plays critical roles in bone for- mation and osteoblast differentiation [23]. We discovered a strong association between the number of STRO-1+ cells and the genes expressing BMPR1A, MSX2, Runx2, and ALP (Figure 3). In addition, we found a correlation between the number of STRO-1+ cells and ALP activity in early passage BMSCs but not in later passages. This leads us to suspect that the culture of human BMSCs can result in the predominance of different cell phenotypes. Since we did not measure STRO-1+ cells in P3 this cannot be confirmed.

This study was the first attempt to test the responses of BMSCs to a panel of osteogenic drugs. We found that the response of BMSCs to a panel of osteogenic drugs was highly diversified. Though BMPs might play a critical role in enhancing implant osseointegration [24], the response of human BMSCs to BMP-2 was quite varied. Current clinical studies have shown variable

success rate of recombinant BMP in the treatment of fracture repair and nonunion [25]. As compared to results observed in animal models, BMPs are relatively inefficient in inducing human BMSCs to undergo osteogenesis [26]. We found that BMP-2 stimulated the BMPR-pSMAD1/5 signaling pathway but this did not correlate with ALP activity in these cultures, suggesting there is a distinctive molecular mechanism or sig- naling pathway for its osteogenic action.

Bisphosphonates are widely used as therapeutic agents in bone disorders including osteoporosis due to their osteoclast inhibitory effect [27]. Recent data show that bisphosphonates may also induce bone-building by stimulating osteoblast activ- ity [28], including an increase in BMP-2 gene expression [29]. In animal models, the local application of bisphosphonate im- proves periprosthetic bone quality and osseointegration [28]. Wilkinson et al. [30] found that bisphosphonate therapy re- duced femoral calcar bone loss and bone turnover in a group of patients over 2 years after THA. von Knoch et al. [29] evaluat- ed the effects of bisphosphonates on the proliferation and os- teogenic differentiation of human BMSCs isolated during THA. Cultured BMSCs were treated with or without a bisphospho- nate (alendronate, risedronate, or zoledronate) and analyzed over 21 days. Bisphosphonate treatment enhanced BMSC pro- liferation and initiated osteoblastic differentiation. However, the drug responses were highly diversified among the differ- ent donors [29]. The molecular mechanism for the variation of donor responses has not been fully investigated. Our data demonstrated that bisphosphonate (etidronate) treatment in- creased ALP activity in 3 of 11 patient cultures through a pS- MAD-independent signaling pathway. Additionally only one of eleven patient cultures responded to etidronate with an in- crease in BMPR1a expression.

It is well established that DX increases ALP activity in cultured BMSCs. Diefenderfer et al. [31] reported that osteogenesis can be induced by DX (100 nM). Our results confirmed this in 70% of BMSC samples collected from THA patients during surgery. In agreement with these findings, we demonstrated that the continuous presence of DX was an absolute requirement for in vitro osteogenesis in human BMSCs. All cultures treated with DX exhibited Alizarin Red staining for calcium phosphate pro- duction after 21 days of continuous treatment. Because DX is inexpensive and its clinical properties are well understood, it could provide a convenient, safe alternative to BMP as a means of inducing osseointegration in THA patients.

Taken together, we found that the response of BMSCs to osteo- genic drugs was highly diversified in these 13 THA patients. The bone marrow microenvironment is a complex, three-di- mensional structure composed of many cell types and abun- dant extracellular matrix. More efforts are needed to elucidate how the bone marrow composition is related to the osteogenic potential (drug response and ALP activity) and whether this would allow prediction of a clinical outcome. One critical av-

enue for further investigations is to define the number and quality of osteoblast precursor cells among BMSCs. It remains unclear whether the cell surface marker STRO-1 identifies a population of osteoblast precursor cells in the bone marrow. Additional markers, such as BMPRs, may be required to define the osteoblast precursor cells in the bone marrow.

Data generated from this study demonstrated individual varia- tions in response of BMSCs to a panel of osteogenic drugs. The lack of further clinical outcome measurements, such as com- puted tomography (CT) or dual-energy X-ray absorptiometry (DXA), to assess bone changes is a potential limitation.

Data generated from this study is important, because a better understanding of an individual patient’s bone marrow profile and response sensitivity to different osteogenic drugs may help clinicians to predict and prevent post-THA implant failure due to the disruption of osseointegration. A comprehensive “bone marrow osteogenic profile” analysis will help clinicians and biomedical engineers to provide patient-specific implant surface-based osteogenic drug delivery strategies [32-34].

Acknowledgments

The authors wish to thank Dianna Fan, MSs for data collection and literature reviews.

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