Comparison of Mesenchymal Stem Cells (Osteoprogenitors) Harvested From Proximal Humerus and Distal Femur During Arthroscopic Surgery

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1 Original Article With Video Illustration Comparison of Mesenchymal Stem Cells (Osteoprogenitors) Harvested From Proximal Humerus and Distal Femur During Arthroscopic Surgery Knut Beitzel, M.A., M.D., Mary Beth R. McCarthy, B.S., Mark P. Cote, D.P.T., Thomas J.S. Durant, P.T., David M. Chowaniec, B.S., Olga Solovyova, B.S., Ryan P. Russell, M.A., Robert A. Arciero, M.D., and Augustus D. Mazzocca, M.S., M.D. Purpose: The aim of this study was to examine the relations between age, gender, and number of viable mesenchymal stem cells (MSCs) in concentrated bone marrow (BM) obtained from the proximal humerus and distal femur during arthroscopic surgery. Methods: BM was aspirated from either the proximal humerus (n ¼ 55) or distal femur (n ¼ 29) during arthroscopic surgery in 84 patients ( years). MSCs were obtained from fractionated bone marrow after a 5-minute spin at 1,500 rpm. Volume of BM and number of nucleated cells (NCs) were calculated, and samples were cultured for 6 days, after which point colony-forming units (CFUs) were quantified and fluorescence-activated cell sorting (FACS) analysis was performed. Simple linear regression was used to explore relations between age, gender, volume of aspirated BM, and MSCs per milliliter. Results: BM aspirations yielded a mean quantity of ml. After centrifugation, nucleated cells/ml of concentrated BM were harvested. The proximal humerus provided , and the distal femur, , for an overall MSCs/mL of concentrated BM (proximal humerus: , distal femur: ). Values did not significantly differ by age, gender, or donor site. Conclusions: Arthroscopic aspiration of bone marrow from the proximal humerus and distal femur is a reproducible technique and yields reliable concentrations of MSCs. The use of an intraoperative concentration method resulted in consistent amounts of MSCs in all clinically relevant age groups without a significant drop of the number of isolated MSCs. Clinical Relevance: Human MSCs derived from concentrated bone marrow aspirate are a promising biological addition that may have practical use in the future of soft tissue augmentation. Arthroscopic techniques for bone marrow aspiration that do not require an additional surgical site for aspiration (e.g., iliac crest) or a second operative procedure may facilitate future use of MSCs in arthroscopic surgery. Autologous human mesenchymal stem cells (MSCs), alternatively referred to as connective tissue progenitor cells, are an example of a biological From the Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, Connecticut, U.S.A. The authors report the following potential conflict of interest or source of funding in relation to this article: Supported by direct funding and material provided by Arthrex (Naples, FL) to the University of Connecticut Health Center/New England Musculoskeletal Institute. The company had no influence on study design, data collection, interpretation of the results, or the final manuscript. A.D.M. receives research support from Arthrex and is also a consultant for Arthrex. Received September 14, 2011; accepted August 22, Address correspondence to Augustus D. Mazzocca, M.S., M.D., Department of Orthopaedic Surgery, University of Connecticut, Farmington, CT 06034, U.S.A. mazzocca@uchc.edu Ó 2013 by the Arthroscopy Association of North America /11576/$ addition that may have practical use in the future of soft-tissue augmentation. 1-6 Human MSCs are defined as multipotent, nonhematopoietic stromal cells that have the potential to undergo self-renewal (i.e., ability to differentiate into bone, tendon, cartilage, and ligament). Introduction of MSCs into a site of injury may have practical benefit in enhancing the healing process. Further, local proliferation of growth factors and cytokines occurring in vivo may increase multipotency potential of MSCs, thereby improving the healing response at the site of injury. Treatment using MSCs has historically involved the transplantation of cells into patients, through either local delivery or systemic infusion (e.g., hematopoietic stem cell transplantation, which has been used extensively in the field of oncology). 7 Clinical application of MSCs in orthopaedic procedures is limited to use in bone fracture repair and osteonecrosis There are surgical techniques in orthopaedic medicine that do not Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 29, No 2 (February), 2013: pp

2 302 K. BEITZEL ET AL. involve harvesting or concentrating bone marrow yet are designed to use MSCs to aid the repair process. Cartilage repair techniques such as microfracture operate on the premise that stimulating bone marrow may introduce MSCs into the site of the defect, allowing tissue ingrowth through cell differentiation. 12 MSCs may be of similar use in procedures involving fixation of tendon to bone, such as rotator cuff repair or placement of a tendon graft in a bone tunnel, as is the case in anterior cruciate ligament (ACL) reconstruction. However, to date research supporting the use of MSCs to facilitate tendon-to-bone healing is limited to basic science investigation. 13 Traditionally, mesenchymal stem cells have been obtained through isolation of bone marrow aspirated from the iliac crest. 8,14 Methods for harvesting MSCs from peripheral blood have also been described. Systemic treatment of peripheral blood with granulocytee macrophage colony-stimulating factors has been used to increase the production of stem cells for bone marrow transplantation. 15 Although these methods have been well documented, they are not very suitable for use during arthroscopic rotator cuff repair or ACL reconstruction as they require an additional procedure and increase patient morbidity. We have recently described aspiration techniques for the proximal humerus and distal femur that allow the surgeon to harvest bone marrow during arthroscopic surgery in a reproducible manner without significant increases in operating time, patient complications, or compromise in efficiency or sterility (see Video 1, available at 16,17 The wide age range of patients undergoing rotator cuff repair or ACL reconstruction raises a question regarding the relation between age and numbers of MSCs obtained from the proximal humerus and distal femur. Studies on age-related changes in MSC concentration have been variable, with results showing either a decrease, increase, or no change in cell numbers with increasing age. 8,18-21 These studies used traditional donor sites (i.e., pelvis and lumbar spine) for aspiration and included a large spectrum of subjects covering several decades of life. Patients electing for rotator cuff repair (older demographic) or ACL reconstruction (younger demographic) represent a distinct distribution of ages that does not include patients in the very early or very late stages of life. As such, exploration of age-related effects on MSCs obtained from the proximal humerus and distal femur in a sample representative of this population is needed to elucidate whether a relation similar to that reported previously exists. The purpose of this study was to examine the relation between age, gender, and number of MSCs in concentrated bone marrow from 2 different aspiration sites, proximal humerus and distal femur, in a group of patients undergoing arthroscopic rotator cuff repair or ACL reconstruction. Our hypothesis was that the number of MSCs obtained from concentrated bone marrow, aspirated from the proximal humerus and distal femur, would remain consistent throughout the age range of the population commonly undergoing surgery for rotator cuff or ACL reconstruction. Methods Patients The institutional review board approved the experimental protocol for this prospective study (IRB No ). From January 2006 to December 2010, 101 patients ( years) underwent bone marrow aspiration from either the proximal humerus or distal femur during arthroscopic surgery. Patients were eligible for inclusion if they were older than 20 and if they underwent rotator cuff surgery or ACL reconstruction within the Department of Orthopaedic Surgery of the University of Connecticut Health Center. Excluded were patients in vulnerable populations, such as prisoners; pregnant women; individuals who had a history of infectious disease, including hepatitis, human immunodeficiency virus, and diabetes; and those who had been exposed to therapeutic radiation (e.g., cancer treatment) and or had bone marrow (BM)- derived illnesses (e.g., leukemia), because of the potential of altered bone marrow status. Bone Marrow Aspiration Bone marrow was aspirated from the proximal humerus in arthroscopic rotator cuff surgery according to a previously published method. 16 A 14-gauge needle fit with a 60-mL syringe containing 1 ml of 1,000 U of preservative-free sodium heparin and 9 ml of saline was inserted 2.5 to 3 cm into the medial aspect of the greater tuberosity (Bone Marrow Aspiration Kit, Arthrex, Naples, FL). The tunnel created was later used to insert the first medial suture anchor. In a standardized method of aspiration, the surgeon pulled back on the syringe to maximize suction for 60 seconds, allowing BM to flow into the syringe. The surgeon aimed on collecting a minimum of 15 ml of aspirated BM; however, if more could be aspirated within the 60 seconds, a larger amount would be collected (Fig 1A). BM was collected in a comparable manner in arthroscopic ACL surgery (Fig 1B). The aspiration needle was placed arthroscopically in the superior lateral or medial aspect of the femoral notch and inserted parallel to the longitudinal axis of the femur for 3 cm. Then BM was aspirated with the same procedure used in the shoulder joint. No tourniquet was used when aspirating BM. Isolation of Mesenchymal Stem Cells The previously described intraoperative method of MSC isolation was used. 16 After determination of the

3 SHOULDER/KNEE OSTEOPROGENITOR COMPARISON 303 phenol red-free a Minimum Essential Medium (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA), and 0.1% penicillin/streptomycin (Invitrogen). To ensure that sterility was maintained, all cultures were checked daily for contamination. To elucidate whether MSCs were able to differentiate into bone, cells were grown in osteogenic stem cell medium (OSCM), which contains 50 mg/ml ascorbic acid, 4mMb-glycerol phosphate, and 10 8 M dexamethasone (Sigma). 22 To quantify the number of MSCs in the sample capable of osteogenesis, colony-forming units (CFUs) expressing alkaline phosphatase in culture were counted. After 7 days of culture, 8 or more cells in a cluster were counted and defined as CFUs. 23 Fig 1. (A) Intraoperative view of arthroscopic bone marrow aspiration from the proximal humerus in rotator cuff surgery. Note the placement of the aspiration cannula in the anterolateral portal. (B) Intraoperative view of arthroscopic bone marrow aspiration from the distal femur in arthroscopic ACL reconstruction surgery. The aspiration needle is placed in the medial portal with the arthroscope in the standard lateral portal. The surgeon used a 60-mL syringe for aspiration and pulled for optimal suction to allow bone marrow to be aspirated for approx 60 seconds for both aspiration sites. overall volume of BM aspirated, a surgical technician carefully overlaid the aspirated BM onto a 17.5% sucrose gradient in a 50-mL conical tube (Fisher Scientific, Agawam, MA). The tube was capped, passed off the surgical field, and centrifuged on site in the operating room for 5 minutes at 1,500 rpm (205g). MSCs in the fractionated middle layer were then extracted using the same aspiration needle used to obtain the BM. 16 The volume aspirated was determined and taken to the laboratory for culture. Cell Culture and Quantification of Colony-Forming Units The nucleated cells harvested from the fractions were counted and plated on 100-mm Primaria dishes (BD Laboratories, Franklin Lakes, NJ) at a concentration of cells/9.6 cm 2 in control medium containing Flow Cytometric Analysis Fluorescence-activated cell sorting (FACS) was used to ensure that cells obtained from the proximal humerus had surface markers characteristic of stem cells. Cells were grown in control medium, trypsinized in 0.25% trypsin/edta at confluence, rinsed, and centrifuged. The pellet was resuspended in staining buffer containing 1% human serum, 1% bovine serum albumin, and 1% fetal bovine serum in phosphate-buffered saline. Cells were incubated with either phycoerythrin or fluorescein isothiocyanate antibodies, washed with staining buffer, and analyzed using a FACS Calibur (BD Biosciences, San Jose, CA). To identify MSCs, phycoerythrin-conjugated mouse monoclonal anti-cd73 immunoglobulin G (IgG), anti-cd90 IgG, and fluorescein isothiocyanate-conjugated anti-mouse CD45 monoclonal IgG were obtained from BD Biosciences. These were chosen in correlation with the suggestions of the International Society for Cell Therapy. 24,25 All antibodies were reactive against human antigens. Testing with negative and positive controls confirmed the specificity of these antibodies. Statistical Analysis Data were stratified into age ranges by decade for comparison of groups (21 to 30, 31 to 40, etc.). Means were compared between groups. One-way analysis of variance (ANOVA) was used to compare means for age groups (decades). If significant findings were detected, the Bonferroni test was used for post hoc analysis. For comparison of gender and donor site, the t test was used. A P <.05 was used to determine statistical significance. Linear regression analysis was performed, and the coefficient of determination (r 2 ) was determined as a robust measure for correlation. All statistical analyses were performed using SPSS (SPSS, Chicago, IL). Results Patients Eighty-four patients ( years) met the inclusion and exclusion criteria and were included in

4 304 K. BEITZEL ET AL. Table 1. Distribution of Number of Patients, Gender, and Age by Aspiration Site Number Age (yr) Aspiration site Female Male Female Male Proximal humerus Distal femur Overall the study. The mean age of the patients undergoing aspiration of BM from the distal femur ( years) was lower than that of the patients undergoing aspiration from the proximal humerus ( years). However, this difference was not significant (P ¼.095). The prevalence of gender, site of BM aspiration, and age distribution is outlined in Table 1. Bone Marrow Aspiration Bone marrow was successfully aspirated in all 84 patients. The overall amount of BM aspirated was ml. After 5 minutes of centrifugation, the overall volume of concentrated BM was ml, including nucleated cells/ml of concentrated BM. At the proximal humerus, the mean volume of BM aspirated was ml with a mean of nucleated cells/ml of concentrated BM. At the distal femur, the volume of BM aspirated was ml with a mean of nucleated cells/ml of concentrated BM. Comparison of volume of BM aspirated, concentrated volume of BM, or overall number of nucleated cells per milliliter of concentrated BM yielded no significant differences between the age groups (Fig 2A), between male and female patients (Fig 2B), or between donor sites (Fig 2C). Cell Culture and Quantification of Mesenchymal Stem Cells Colony-forming units were counted after 7 days of cell culture to evaluate the number of MSCs with osteogenic potential (ALK staining). Overall, CFUs grew per 10 6 nucleated cells. This resulted in MSCs/mL of concentrated BM. At the aspiration site in the proximal humerus, nucleated cells/ml of BM were isolated, and of these, MSCs/mL were cultured. For the distal femur, there were nucleated cells/ml of BM and MSCs/mL, respectively. There were no significant significances in the concentration of MSCs between age groups (Fig 3A), genders (Fig 3B), or donor sites (Fig 3C). Regression Analysis Linear regression analysis was performed to evaluate the correlation between age of patients and nucleated cell count, as well as MSCs per milliliter of aspirated BM for the overall population, according to gender and aspiration site. For the overall number of nucleated cells per milliliter of BM after centrifugation, r 2 ¼ 0.001, and for the amount of MSCs per milliliter of concentrated BM, r 2 ¼ 0.003, respectively. At the aspiration site of the proximal humerus, r 2 ¼ for nucleated cells per milliliter of BM and for MSCs per milliliter of BM. At the distal femur, r 2 ¼ 0.1 and 0.07, respectively. When the numbers were compared by gender, only low correlations were shown (nucleated cells/ml: r 2 male ¼ 0.01, r 2 female ¼ 0.12; MSCs/mL: r 2 male ¼ 0.00, r 2 female ¼ 0.047). Evaluation of the correlation of aspirated volume of BM with resulting cell concentrations yielded r 2 ¼ for concentration of nucleated cells and r 2 ¼ for concentration of resulting MSCs. Flow Cytometric Analysis The FACS analysis showed that MSCs grown in control medium were positive for CD73 and CD90 and simultaneously negative for CD45, in addition to their potential to form colonies, differentiate into different cell lines, and adhere to plasticda fourth additional characteristic criterion of adult MSCs. 25 Discussion The purpose of this study was to investigate the relation between donor age/sex and concentration of viable MSCs isolated from BM aspirated from the proximal humerus or distal femur during arthroscopic surgery. Current reports concerning MSC concentration and donor age within the specific age range of surgical candidates for rotator cuff repair and ACL reconstruction are limited, and indicate variable results. 19,20 The results of the present study suggest that arthroscopic aspiration of BM from the proximal humerus and distal femur is a reliable procedure yielding sufficient volumes of BM and consistent isolation of MSCs in all clinically relevant age groups. 16 Additional analysis exploring the effect of gender did not reveal a significant difference between male and female donors. Our findings are consistent with previous investigations, which have reported either the absence of a correlation or a modest decrease in cell viability with increasing age. 8,18,21 In our study population, we were able to isolate MSCs from patients older than 60 without an increased failure rate as described by Bertram et al. in his study of 32 patients. 18 Nishida et al. evaluated 49 female patients ranging from 4 to 88 years of age. The most dramatic dropoff in osteogenic MSC concentration in their study occurred in the first 2 decades. 20 We did not include patients younger than 20 because our study sought to include patients in an adult age range generally undergoing ligament or tendon surgery. Our target age demographic may also be the reason our compared results differed from those of Muschler et al. 19 Their population was distributed more

5 SHOULDER/KNEE OSTEOPROGENITOR COMPARISON 305 Fig 2. Number of nucleated cells (millions) per milliliter of concentrated bone marrow (BM) by (A) linear regression of age distribution (years); (B) gender; and (C) donor site. toward elderly patients, and in addition, only 2 ml of BM was aspirated from varying areas of the iliac crest. Although differences in MSC concentration exist between newborn infants and adult populations, trends throughout the adult population may be skewed by inclusion of the very young age demographic. 24 Trends observed in this study are relative to the target population, which is commonly seen in sports medicine orthopaedic practice. The distal femur and proximal humerus were chosen as donor sites for this study because of their accessibility during arthroscopic rotator cuff repair or ACL reconstruction. Although aspiration of BM from the iliac crest is still considered the gold standard, 11 the investigation of sites accessible within the primary surgical field seemed warranted to minimize potential morbidity for the patient. These risks include increased pain, infection, postoperative bleeding, and neurovascular damage. 26 Both sites of aspiration yielded consistent volumes of BM and nucleated cells. Aspirate obtained from the proximal humerus yielded a higher concentration of MSCs than BM obtained from the proximal femur;

6 306 K. BEITZEL ET AL. Fig 3. Number of colony-forming units (CFUs) per milliliter of concentrated bone marrow (BM) by (A) linear regression of age distribution (years); (B) gender; and (C) donor site. however, this trend was not statistically significant. 11 Similar variations in MSC concentration have also been shown between the iliac crest and vertebral spine. We also observed high interindividual variations in number of nucleated cells and concentrations of MSCs in BM aspirates, which is consistent with previous studies of BM aspiration. 11,18,27,28 It is difficult to directly compare amounts of isolated cells between different previously published studies, because differences in methods of isolation, culture times until CFU definition, and differentiation media used may result in very diverse outcomes. Hernigou et al. (60 patients; mean age, 40 years) were able to isolate 2,579 1,121 MSCs/mL of concentrated BM with the use of an automated cell separator. 8 The gross difference between their number and the current results may additionally be attributed to the fibroblastic differentiation line used in their study. 8 McLain et al. (42 patients; mean age, 59 years) were able to isolate a more comparable number of MSCs when matching the amounts isolated from the iliac crest ( MSCs/mL of concentrated BM) and vertebral body ( MSCs/mL); however, they found significant differences when comparing the harvesting

7 SHOULDER/KNEE OSTEOPROGENITOR COMPARISON 307 sites. However, in 2009, the same study group harvested BM from the iliac crest and concentrated it (13 patients; mean age, 59 years), yielding MSCs/mL of concentrated BM. 28 For standardization of the procedure, the aspiration process was completed after approximately 60 seconds. This resulted in variable volumes of aspirated bone marrow (>15 ml). Previous literature suggested that the first 10 to 20 ml of aspirated BM contains a higher concentration of MSCs and larger volumes are more highly diluted because of the increased content of peripheral blood. 27 Additionally, MSCs are believed to adhere to the microstructure of the trabecular bone. On the basis of similar observations, Muschler et al. recommended dividing the aspiration process into repetitive aspirations of smaller volumes (2 ml) from different locations (e.g., turning the aspiration needle 45 ). 27 Because of the specific nature of arthroscopic surgery, we do not recommend various insertions of the aspiration needle in the procedure. However, the surgeon should be aware of this dilution factor and therefore slightly change the depth of insertion or turn the needle to obtain an optimal aspirate. For consistency with our own research as well as previously published data, cells were cultured in an osteogenic medium to examine whether differentiation into osteogenic cells was possible. 16,19,20,25 Alkaline phosphatase (ALK) staining-revealed cultured cells exhibited cellular characteristics consistent with bone, and FACS analysis added further confidence to the fact that the isolated cells were multipotential MSCs. 25,29 CD73, CD90, and CD45 were chosen as markers for this study, whereas CD105 could not be used because it was not available to our study group for the current study. The results support the presence of MSCs in bone marrow, thereby allowing the cell population to be defined in a manner consistent with previously published literature. 19,20,25,29,30 Limitations There are several limitations to this study. Differentiation into additional cell types would have further verified MSC lineage. 24,30 As recommended by the International Society for Cellular Therapy (ISCT), 4 commonly known criteria (cell adherence to culture flasks, growth into colony-forming units, FACS analysis, differentiation into osteogenic cell line) were used to define the presence of MSCs in our cell population. 16,19,20,25,29,30 Another limitation is that the ages of our patients were not distributed equally, which might have resulted in more significant findings. However the distribution of ages in the present study was comparable to that of the patient population electing to undergo arthroscopic ACL reconstruction or rotator cuff repair. Additionally, not all patients who underwent aspiration of BM from either the distal femur or proximal humerus have been included in the final study population. Approximately 4% of all aspirates obtained during the time frame of the study were not included in this analysis because of either missing assays or contamination. Conclusions Arthroscopic aspiration of bone marrow from the proximal humerus and distal femur is a reproducible technique and yields reliable amounts of mesenchymal stem cells. The use of an intraoperative concentration method resulted in consistent amounts of mesenchymal stem cells in all clinically relevant age groups without a significant drop in the number of isolated mesenchymal stem cells. References 1. Awad HA, Butler DL, Boivin GP, et al. Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng 1999;5: Gulotta LV, Kovacevic D, Packer JD, Deng XH, Rodeo SA. Bone marrow-derived mesenchymal stem cells transduced with scleraxis improve rotator cuff healing in a rat model. Am J Sports Med 2011;39: Chong AK, Ang AD, Goh JC, et al. 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