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The Journal of Bone and Joint Surgery 79:1699-1709 (1997)
© 1997 The Journal of Bone and Joint Surgery, Inc.

Aspiration to Obtain Osteoblast Progenitor Cells from Human Bone Marrow: The Influence of Aspiration Volume*

GEORGE F. MUSCHLER, M.D.{dagger}, CYNTHIA BOEHM, B.S.{dagger} and KIRK EASLEY, M.S.{dagger}, CLEVELAND, OHIO

Investigation performed at The Cleveland Clinic, Cleveland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone marrow contains osteoblast progenitor cells that can be obtained with aspiration and appear to arise from a population of pluripotential connective-tissue stem cells. When cultured in vitro under conditions that promote an osteoblastic phenotype, osteoblast progenitor cells proliferate to form colonies of cells that express alkaline phosphatase and, subsequently, a mature osteoblastic phenotype. We evaluated the number of nucleated cells in bone-marrow samples obtained with aspiration from the anterior iliac crest of thirty-two patients without systemic disease. There were nineteen male patients and thirteen female patients; the mean age was forty-one years (range, fourteen to seventy-seven years). The prevalence and concentration of the osteoblast progenitor cells also were determined, by placing the bone-marrow-derived cells into tissue-culture medium and counting the number of alkaline phosphatase-positive colony-forming units. In order to assess the effect of aspiration volume, two sequential experiments were performed. In the first experiment, aspiration volumes of one and two milliliters were compared. In the second experiment, aspiration volumes of two and four milliliters were compared. The mean prevalence of alkaline phosphatase-positive colony-forming units in the bone-marrow samples was thirty-six per one million nucleated cells (95 per cent confidence interval, 28 to 47); a mean of 2400 alkaline phosphatase-positive colony-forming units was obtained from a two-milliliter aspirate. There was a significant difference among the patients with respect to the number of alkaline phosphatase-positive colony-forming units in these bone-marrow samples (p < 0.001). Seventy per cent of this variation in the prevalence was due to variation among patients, and 20 per cent was due to variation among aspirates. The number of alkaline phosphatase-positive colony-forming units in the aspirate increased as the aspiration volume increased. However, contamination by peripheral blood also increased as the aspiration volume increased. An increase in the aspiration volume from one to four milliliters caused a decrease of approximately 50 per cent in the final concentration of alkaline phosphatase-positive colony-forming units in an average sample. CLINICAL RELEVANCE: On the basis of these data, we recommend that, when bone marrow is obtained with aspiration for use as a bone graft, the volume of aspiration from any one site should not be greater than two milliliters. A larger volume decreases the concentration of osteoblast progenitor cells because of dilution of the bone-marrow sample with peripheral blood. We estimate that four one-milliliter aspirates will provide almost twice the number of alkaline phosphatase-positive colony-forming units as will one four-milliliter aspirate. In addition, these data confirm that humans differ significantly from one another with respect to the cellularity of bone marrow and the prevalence of osteoblast progenitor cells. Additional studies are necessary to determine if the number or prevalence of alkaline phosphatase-positive colony-forming units in bone marrow is a determining factor in the efficacy of an autogenous bone or bone-marrow graft and to ascertain how the number and function of alkaline phosphatase-positive colony-forming units may change as a function of factors such as age, menopausal status, and selected diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone-grafting is widely used to treat fractures and non-unions, to induce therapeutic fusion, and to fill segmental bone defects. Autogenous cancellous bone is currently the most effective graft material. However, several synthetic and tissue-derived materials are now available for bone-grafting, and many others are under development. These materials include processed type-I collagen, calcium-phosphate ceramics, and extracted or recombinant protein-adhesion molecules and growth factors.

In selected settings, these alternative materials may be as efficacious as autogenous cancellous bone11,17-20,27,43,44,64. When this is the case, use of a synthetic graft material may preclude the need for autogenous bone and thus may protect the patient from the morbidity and costs associated with autogenous bone grafts.

However, the ability of these materials to generate bone-healing depends totally on the presence of a sufficient number of competent osteoblast progenitor cells in the graft site. Since the population of local progenitor cells may be deficient in many clinical settings—for example, the sites of non-united bone defects, osteonecrosis, previous radiation therapy, or previous infection—the effectiveness of these new materials may be suboptimum unless osteoblast progenitor cells are included in the graft.

Osteoblast progenitor cells are found in several tissue locations, including the periosteum21,47, the peritrabecular connective tissue3,32,51, and possibly the vascular pericyte9,10,21. However, the most abundant and most readily available source of osteoblast progenitor cells is bone marrow.

Bone marrow has been found to contain osteoblast progenitor cells in many animal studies2,6,13,18-21,23-26,29,30,40,54,59-63. Numerous investigations of whole-marrow transplants in small animals and of bone marrow aspirated from dogs have demonstrated that bone marrow is an effective bone graft both by itself and in combination with other materials12,33,35,36,45,46,48-50,55,56,59-61,65.

Clinical studies also have suggested that aspirated bone marrow has value as a bone-graft material. Connolly et al.16 reported that percutaneous injections of 100 to 150 milliliters of non-heparinized bone marrow were successful in the treatment of eighteen of twenty tibial non-unions when combined with either use of a cast or fixation with an intramedullary nail. The bone-marrow grafts contained a mean of approximately 3 x 109 nucleated cells and were obtained from the posterior iliac crest in three to five-milliliter aspirates16. Healey et al.31 reported that five of eight non-unions in patients who had cancer united after a mean of 1.5 percutaneous injections of fifty milliliters of non-heparinized bone marrow obtained from the iliac crest in three to six-milliliter aspirates. Connolly et al.15 also suggested that intraoperative processing of bone marrow in order to concentrate the osteoblast progenitor cells may improve its efficacy as a bone graft. They reported that the osteoblastic activity in a diffusion chamber was increased by concentration of cells with a low density (less than 1.075 grams per milliliter) that were obtained from the whole marrow of rabbits.

Recognizing the potential biological value of bone marrow, many surgeons have begun to use it as an adjuvant to allografting. However, we are not aware of any randomized clinical trial that has documented the effectiveness of bone-marrow grafts in this setting, and few data are available to guide the surgeon in selecting the optimum technique for bone-marrow aspiration.

If the value of bone marrow as a graft material is to be evaluated objectively, many important issues must first be addressed, including the number of osteoblast progenitor cells that can be obtained with aspiration of human bone marrow, the ways in which the aspiration technique can be optimized, the best site for performing aspiration, and any limitations imposed by factors such as age or disease that may make some patients unsuitable candidates for aspiration. We also must investigate the possibility that the processing of bone marrow before transplantation may improve the outcome of bone-healing. For example, processing may allow the surgeon to increase the number of osteoblast progenitor cells transplanted in a graft or to selectively remove components of bone marrow that may inhibit the bone-healing response. Whether available matrix materials enhance the performance of bone marrow in specific clinical settings, and vice versa, also must be determined. Finally, we must define the clinical settings in which bone-marrow grafting alone is as effective as the use of autogenous bone without the additional expense of alternative grafts.

Many controlled clinical studies will be necessary to evaluate the role of bone marrow as a material for bone-grafting. The design and execution of these studies will require a clear understanding of the variables and variability associated with obtaining osteoblast progenitor cells from human bone marrow. Protocols will be needed to standardize aspiration techniques that are most likely to optimize the number and concentration of osteoblast progenitor cells that are obtained. The current study had two specific goals: to determine the numbers of alkaline phosphatase-positive colony-forming osteoblast progenitor cells in bone-marrow aspirates obtained during elective orthopaedic operations, and to define how the number and concentration of osteoblast progenitor cells are influenced by aspiration volume.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 

Experimental Design
Patients who were scheduled to have an elective orthopaedic procedure performed by one of us (G. F. M.) were recruited for the study. All patients were fully informed with respect to the rationale of the study and the associated risks, in accordance with a protocol and a signed consent form approved by the Institutional Review Board of The Cleveland Clinic Foundation. Patients who were more than three years of age were eligible. Patients who had an active infection were excluded.

Two sequential experiments were performed. In Experiment 1, aspiration volumes of one and two milliliters were compared. In Experiment 2, volumes of two and four milliliters were compared.

The design of each experiment was the same. Each patient had percutaneous aspiration of bone marrow from both the left and the right anterior iliac crest after induction of anesthesia. Samples were obtained from four separate sites on each iliac crest. In order to minimize the influence of potential differences related to the site or side, the technique was alternated: for example, a one, two, one, two-milliliter sequence was used on the left, followed by a two, one, two, one-milliliter sequence on the right.

Patient Population for Experiment 1
Sixteen consecutive patients were recruited for the comparison of the one-milliliter aspiration volume with the two-milliliter volume. The mean age of the patients was thirty-nine years (range, nineteen to seventy-seven years). There were nine male patients and seven female patients. Six patients had excision of a neoplasm (an aneurysmal bone cyst, lipoma, osteochondroma, lymphoma, chondrosarcoma, or osteosarcoma) of an extremity, five had treatment of a tibial non-union, and five had a hip or knee arthroplasty for the treatment of osteoarthrosis.

Patient Population for Experiment 2
Seventeen consecutive patients were recruited for the comparison of the two-milliliter aspiration volume with the four-milliliter volume. The mean age of the patients was forty-three years (range, fourteen to seventy-one years). There were eleven male patients and six female patients. Seven patients had a hip or knee arthroplasty for the treatment of osteoarthrosis, five had treatment of a non-union (three, of a tibial fracture; one, of a humeral fracture; and one, of a radial fracture), three had excision of a neoplasm (a desmoid tumor, osteochondroma, or lipoma), one had release of a hip contracture, and one had removal of a hip compression screw.

One patient who had a tibial non-union contributed bone-marrow aspirates to both experiments, six months apart. Therefore, thirty-two patients contributed to the study over-all.

Aspiration Technique
After anesthesia had been induced and before the operative procedure had begun, both anterior iliac crests were prepared with use of sterile technique. A two-millimeter stab incision was made parallel to the Langer lines in the skin, approximately five centimeters posterior and lateral to the anterior superior iliac spine. A bone-marrow aspiration needle (Lee-Lok, Minneapolis, Minnesota) was advanced into the intramedullary cavity at a site approximately two centimeters directly posterior to the anterior superior iliac spine and one to two centimeters distal to the iliac crest. The obturator was removed, and a ten-milliliter syringe containing one milliliter of heparinized (1000 units per milliliter) normal saline solution was fixed to the needle. Negative pressure was established by drawing the plunger back to approximately the six-milliliter marker until marrow began flowing into the syringe; the pressure then was reduced, and the appropriate volume of marrow was collected for three to six seconds. The syringe was detached and was inverted several times to ensure complete mixing. The entire sample then was suspended in twenty milliliters of alpha-minimum essential medium (lot number 11900-073; Gibco, Grand Island, New York) containing twenty units of sodium heparin per milliliter. The three subsequent aspiration sites on each side were spaced at approximately one-centimeter intervals extending posteriorly along the iliac crest.

Assay of Alkaline Phosphatase-Positive Colony-Forming Units
The heparinized bone-marrow suspensions were centrifuged at 1500 revolutions per minute for ten minutes. The buffy coat was pipetted and was resuspended in five milliliters of alpha minimum essential medium. Nucleated cells were counted on a hemocytometer with use of 0.4 per cent trypan-blue exclusion to document viability. Each sample was plated, in six ten-square-centimeter wells, at three different densities (106, 5 x 105, and 105 nucleated cells per well). The culture medium consisted of 90 per cent alpha minimum essential medium, 10 per cent fetal bovine serum (lot number 4M0991; BioWhittaker, Walkersville, Maryland), and dexamethasone (10-8 molar). The plates were incubated at 37 degrees Celsius in 5 per cent CO2. The media were changed on the seventh day40. These are established in vitro conditions that promote the expression of an osteoblastic phenotype40.

On the ninth day, all plates were stained in situ for determination of alkaline-phosphatase activity38. Alkaline phosphatase-positive cell clusters that were two millimeters or more in diameter were counted in each well (Fig. 1). A mean count of alkaline phosphatase-positive colony-forming units was then calculated for each plating density. Wells that were plated at the lowest density (105 cells per well) served as the preferred assay for determination of the mean count. If, for example, the mean count per well at this density was twenty, then the prevalence of alkaline phosphatase-positive colony-forming units in that sample was reported as 200 per one million nucleated cells. However, if the mean was less than ten colonies per well, then wells plated with 5 x 105 nucleated cells were used. Similarly, if wells plated with 5 x 105 nucleated cells also had a mean of less than ten colonies, then wells plated with 106 nucleated cells were used. This protocol provides a counting range from zero to more than 600 alkaline phosphatase-positive colony-forming units per one million nucleated cells. The protocol decreases the likelihood of two potential problems that can occur when such data are collected: undercounting of colonies due to overlap when more than sixty colonies are present in one thirty-five-millimeter-diameter well, and excessive variation in the data when the mean is less than ten colonies per well due to occasional values of zero.



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  		Fig. 1 Photograph of bone-marrow samples, obtained from five separate patients, plated in six-well dishes (one million nucleated cells per well), and maintained in culture medium for nine days. The numbers of alkaline phosphatase-positive colony-forming units that were two millimeters or more in diameter are (left to right) approximately fifteen, thirty, sixty, 120, and 180.

 
In Experiment 1, complete data sets of eight alkaline phosphatase-positive colony-forming-unit assays (four obtained with each [one or two-milliliter] aspiration volume) were collected from thirteen of the sixteen patients. The remaining three patients had incomplete data consisting of four, six, or seven measurements. One patient was excluded from the analysis because the mean counts of alkaline phosphatase-positive colony-forming units were tenfold higher than the mean counts for the remainder of the patients. This patient was a sixteen-year-old girl who had curettage and bone-grafting for the treatment of an aneurysmal bone cyst of the proximal aspect of the femur. (The data for this patient are discussed later.) In aggregate, the data included in the comparison of the one and two-milliliter aspiration volumes were based on 113 measurements from fifteen patients who had seven missing values.

In Experiment 2, complete data sets of eight alkaline phosphatase-positive colony-forming-unit assays (four obtained with each [two or four-milliliter] aspiration volume) were collected from fifteen of the seventeen patients. The remaining two patients had incomplete data: one patient had six measurements, and the other had four. In aggregate, the data included in the comparison of the two and four-milliliter aspiration volumes were based on 130 measurements from seventeen patients who had six missing values.

Statistical Analysis
The distributions of data for both the number of cells and the prevalence of alkaline phosphatase-positive colony-forming units were non-gaussian, exhibiting a positive skew. Non-parametric kernel density estimation was used to describe the shape of the distribution37. A natural logarithm transformation was used to help to alleviate a positive skew. This increased the normality of the data and allowed the use of a repeated-measures analysis-of-variance model for comparison of the aspiration volumes. The independent factors included the identity of the patient and the data from the individual aspirates (eight per patient). Results from the fitting of the statistical model were reported as model-based means, adjusted for the fact that not all patients had eight measurements. Ninety-five per cent confidence intervals were calculated for the mean numbers of alkaline phosphatase-positive colony-forming units and of nucleated cells. Variance components were reported as the proportion of total variability attributable to the patients and the aspirates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 

Description of the Data
The numbers of nucleated cells and the prevalences of alkaline phosphatase-positive colony-forming units in the two-milliliter aspirates from thirty-one patients varied significantly (p < 0.001), both among the patients and among the aspiration samples from the same patient (Figs. 2 and 3). The mean number of nucleated cells for the individual patients ranged from twenty-six to 180 million, and the values from the four assays for Patient 13 ranged from sixty-seven to 320 million. The mean number of alkaline phosphatase-positive colony-forming units per one million nucleated cells for the individual patients ranged from ten to 200, and the values for Patient 21 ranged from sixteen to 460. Descriptive statistics for the nucleated cell-count data and the prevalence data for the alkaline phosphatase-positive colony-forming units from the two-milliliter aspirates, stratified according to the order of aspiration, revealed no trend for differences between the initial and subsequent aspirates (Tables I and II). The geometric mean represents the retransformed model-based mean.



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  		Fig. 2 Graph showing nucleated-cell counts (in millions) in all two-milliliter aspirates from thirty-one patients. The graph illustrates the variation in the nucleated-cell count among patients and among aspirates (dots) from the same patient.

 


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  		Fig. 3 Graph showing the prevalence of alkaline phosphatase-positive colony-forming units (CFU) in all two-milliliter aspirates from thirty-one patients. The graph illustrates the variation in the prevalence among patients and among aspirates (dots) from the same patient.

 

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  		TABLE I DESCRIPTIVE STATISTICS FOR NATURAL LOG NUCLEATED CELLS PER ASPIRATE FOR ALL TWO-MILLILITER ASPIRATES

 

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  		TABLE II DESCRIPTIVE STATISTICS FOR NATURAL LOG OF ALKALINE PHOSPHATASE-POSITIVE COLONY-FORMING UNITS PER MILLION NUCLEATED CELLS FOR ALL TWO-MILLILITER ASPIRATES

 
The total variance in the transformed nucleated cell-count data ({sigma}2 = 1.2751) was attributed (partitioned) to three components: differences among patients (81 per cent), differences among aspirates from the same patient (7 per cent), and variation that could not be explained by the model (12 per cent). For the alkaline phosphatase-positive colony-forming-unit prevalence data, {sigma}2 = 3.3169; the percentage of the variability that was attributable to these three components was 62, 15, and 23 per cent, respectively. Analyses of variability in the two experiments revealed remarkably similar findings, indicating that approximately 70 per cent of the variability was related to differences among patients and that 20 per cent was related to differences among aspirates. The total variance in the alkaline phosphatase-positive colony-forming-unit data in Experiments 1 and 2 was {sigma}2 = 2.4313 and {sigma}2 = 10.3255, respectively, demonstrating the increased variation seen with larger-volume aspirates.

Number and Prevalence of Alkaline Phosphatase-Positive Colony-Forming Units in Human Bone Marrow
The pooling of data from the two-milliliter aspirates obtained from thirty-one patients provided the best estimate of the prevalence of alkaline phosphatase-positive colony-forming units among bone-marrow cells in this patient population. These data indicate geometric means of sixty-six million nucleated cells (95 per cent confidence interval, 55 to 80) and thirty-six alkaline phosphatase-positive colony-forming units per one million nucleated cells (95 per cent confidence interval, 28 to 47), or approximately one alkaline phosphatase-positive colony-forming unit per 28,000 nucleated cells (95 per cent confidence interval, 21,000 to 36,000). Therefore, we estimate that approximately 80 per cent of the cells in a two-milliliter aspirate are bone-marrow-derived and the actual mean prevalence of alkaline phosphatase-positive colony-forming units in native bone marrow for this population is approximately 25 per cent higher, closer to one per 35,000 nucleated cells.

Comparison of Aspiration Volumes

Nucleated Cells
The number of nucleated cells that were obtained with aspiration increased as the aspiration volume increased (Fig. 4). In Experiment 1, the geometric mean was forty million nucleated cells in the one-milliliter aspirates and sixty million nucleated cells in the two-milliliter aspirates, representing a 50 per cent increase (p < 0.001). Similarly, in Experiment 2, the geometric mean was seventy-two million nucleated cells in the two-milliliter aspirates and 100 million nucleated cells in the four-milliliter aspirates, representing a 39 per cent increase (p < 0.001) (Fig. 4).



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  		Fig. 4 Graph showing the mean nucleated-cell count (in millions) according to aspiration volume. Data from the same patient are connected by a solid line. In all except one patient, the mean number of nucleated-cells increased as the aspiration volume increased from two to four milliliters.

 
These data indicate that, as the aspiration volume increases, the fraction of the bone-marrow sample that is composed of peripheral blood increases, resulting in dilution of the sample. Also, as the aspiration volume increases, the concentration of bone-marrow-derived cells decreases quickly. To calculate the fraction and concentration of bone-marrow-derived cells (Tables III and IV), we made two assumptions: first, that the mean nucleated-cell count in peripheral blood for our patient population approximates the mean for a normal population (approximately 7.5 million cells per milliliter), and, second, that the volume of bone-marrow-derived cells contributes minimally to the over-all volume of individual samples since the total volume of ten million nucleated bone-marrow cells is approximately 0.04 milliliter. Therefore, if a mean of forty million cells were obtained from a one-milliliter aspirate, then approximately 32.5 million cells (40 - 7.5 = 32.5), or approximately 81 per cent (32.5 / 40), were bone-marrow-derived. Similarly, the two-milliliter aspirates contained a mean of approximately 77 per cent bone-marrow-derived nucleated cells, and the four-milliliter aspirates contained a mean of approximately 70 per cent bone-marrow-derived nucleated cells.


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  		TABLE III FRACTION AND CONCENTRATION OF BONE-MARROW-DERIVED CELLS IN DIFFERENT ASPIRATION VOLUMES*

 

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  		TABLE IV FRACTION AND CONCENTRATION OF BONE-MARROW-DERIVED CELLS IN SEQUENTIAL FRACTIONS OF AN ASPIRATE*

 

Alkaline Phosphatase-Positive Colony-Forming Units
Mean prevalence data for the alkaline phosphatase-positive colony-forming units were calculated, for all patients, in each of the aspiration volumes (Fig. 5). The estimates of the prevalence of alkaline phosphatase-positive colony-forming units in the low-volume (one and two-milliliter) aspirates were consistent for almost all patients. In contrast, significantly less agreement was found between the estimates for the two and four-milliliter aspirates.



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  		Fig. 5 Graph showing the mean prevalence of alkaline phosphatase-positive colony-forming units (CFU) per million nucleated cells according to aspiration volume. Data from individual patients are connected by a solid line. The data for the one and two-milliliter aspirates are consistent; however, an increase in variation is seen when the two and four-milliliter aspirates are compared. This increase may be related to increased dilution by peripheral blood or to the greater trauma associated with obtaining the four-milliliter aspirates.

 
The number of alkaline phosphatase-positive colony-forming units increased as the aspiration volume increased (Table V). In Experiment 1, the geometric mean was 1451 alkaline phosphatase-positive colony-forming units in the one-milliliter aspirates and 2101 in the two-milliliter aspirates, representing a 45 per cent increase (p = 0.01). In Experiment 2, the geometric mean was 2836 in the two-milliliter aspirates and 3526 in the four-milliliter aspirates, representing a 24 per cent increase (p = 0.23). However, in Experiment 1, the geometric mean concentration of alkaline phosphatase-positive colony-forming units decreased 28 per cent, from 1451 per milliliter in the one-milliliter aspirates to 1051 per milliliter in the two-milliliter aspirates. In Experiment 2, the geometric mean concentration of alkaline phosphatase-positive colony-forming units was 1418 per milliliter in the two-milliliter aspirates and 882 per milliliter in the four-milliliter aspirates, representing a 38 per cent decrease. The rate of dilution of the alkaline phosphatase-positive colony-forming units also was analyzed on the basis of the differences between the one and two-milliliter aspirates in Experiment 1 and the two and four-milliliter aspirates in Experiment 2 (Table VI).


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  		TABLE V NUMBER, CONCENTRATION, AND PREVALENCE OF ALKALINE PHOSPHATASE-POSITIVE COLONY-FORMING UNITS IN DIFFERENT ASPIRATION VOLUMES*

 

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  		TABLE VI NUMBER, CONCENTRATION, AND PREVALENCE OF ALKALINE PHOSPHATASE-POSITIVE COLONY-FORMING UNITS IN SEQUENTIAL FRACTIONS OF AN ASPIRATE*

 
As mentioned earlier, the data on the alkaline phosphatase-positive colony-forming units for one patient, a sixteen-year-old girl who had an aneurysmal bone cyst of the proximal aspect of the right femur, were excluded from the analysis as an outlier. This patient had a mean of 732 alkaline phosphatase-positive colony-forming units per one million nucleated cells and a total of 27,000 alkaline phosphatase-positive colony-forming units per aspirate, representing a tenfold higher prevalence of alkaline phosphatase-positive colony-forming units than was found in the rest of the cohort. Nevertheless, the data from the comparison of the one and two-milliliter aspiration volumes for this patient were consistent with the findings for the rest of the cohort. The mean number of nucleated cells increased from twenty-five million to thirty-seven million, and the mean number of alkaline phosphatase-positive colony-forming units increased from 14,200 to 27,000.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The first goal of this study was to determine the number of osteoblast progenitor cells obtained from bone marrow aspirated from the iliac crests of patients being managed with an elective orthopaedic operation. The number of alkaline phosphatase-positive colony-forming units in a bone-marrow aspirate is determined by two variables: the number of nucleated cells and the prevalence of alkaline phosphatase-positive colony-forming units among the nucleated cells. We know of no previous study in which these types of data were collected from a human population. The current study provides an accurate representation of the variance and distributional properties inherent in such clinical data (Tables I and II).

Several variables may influence the number of cells that are obtained and the prevalence of alkaline phosphatase-positive colony-forming units. For example, the number of nucleated cells increases if the local bone marrow is highly cellular and loosely connected, allowing bone-marrow cells in the marrow space to flow into the aspiration needle. Conversely, the number of nucleated cells decreases if the marrow cavity is hypocellular or relatively fibrotic. In addition, the number and concentration of bone-marrow-derived cells that are obtained are always limited by progressive dilution with peripheral blood. The rate at which this dilution occurs is probably a function of the rate of local blood flow and the number and size of local vessels and sinusoids.

The prevalence of alkaline phosphatase-positive colony-forming units may vary independently of local blood flow or cellularity. An increase in the prevalence or activity of alkaline phosphatase-positive colony-forming units in areas of high bone turnover or recent injury might be expected. However, if pelvic bone marrow is not altered by a local or regional process, the number and function of alkaline phosphatase-positive colony-forming units in bone marrow from the iliac crest should reflect the systemic physiological status of the donor.

Our analysis of the data from the two-milliliter aspirates confirmed that variation in both the number of bone-marrow cells and the prevalence of alkaline phosphatase-positive colony-forming units was high, as we expected. Larger-volume (four-milliliter) aspirates were associated with greater variation. Most of the variation (approximately 60 to 80 per cent) in both the nucleated-cell count and the prevalence of alkaline phosphatase-positive colony-forming units in the two-milliliter aspirates was due to variation among patients. Variation among aspirates accounted for only 10 to 20 per cent of the variation that was observed. This finding supports the concept that the nucleated-cell count and the prevalence of alkaline phosphatase-positive colony-forming units reflect true physiological differences among patients and might be useful in future studies as a tool to investigate the effect of variables such as age, gender, smoking, menopausal status, and selected diseases on the number and function of osteoblast progenitor cells in bone marrow.

We used the number of alkaline phosphatase-positive colony-forming units that formed under these conditions as an estimate of the number of osteoblast progenitor cells57. The literature related to bone-marrow stromal cells in rats supports the concept that the bone-marrow-derived colonies that give rise to alkaline phosphatase-positive colony-forming units are pluripotential connective-tissue progenitor cells that are capable of differentiation into various cell types (fibroblasts, myoblasts, chondrocytes, adipocytes, and osteoblasts)4,5,7,22,28,41,52,53. In support of this assertion, we have shown that a large proportion of alkaline phosphatase-positive colony-forming units go on to express an osteoblastic phenotype. We have characterized primary cultures of human bone-marrow-derived osteoblast progenitor cells with regard to proliferation; alkaline-phosphatase activity; and synthesis of protein, collagen, and osteocalcin38,39. In primary cultures, essentially all colonies exhibit alkaline-phosphatase activity by the fifth day and begin to elaborate a collagenous matrix by the ninth day. By the ninth day, more than 98 per cent of the cells in all colonies express alkaline phosphatase. Confluence is reached at a mean of fourteen to seventeen days. By the twenty-fourth to the forty-fifth day, in the presence of a phosphate source (for example, ß-glycerophosphate or inorganic phosphate), matrix mineralization is observed in a pattern similar to that described by Bellows et al.3 in the bone-marrow stromal cells of rats. Mineralization is associated with synthesis of bone sialoprotein8,42, which we have detected with immunofluorescence. Furthermore, at forty-five days, osteocalcin synthesis can be induced by twenty-four-hour exposure to 1,25-dihydroxyvitamin D3. The concentration of osteocalcin in the media of the culture wells that were plated with one million nucleated cells was consistent with levels that have been found in osteoblastic cell-lines1,14,34,58. However, we have not shown that each colony that is isolated is capable of committing to an osteoblastic phenotype. Therefore, it is possible that some alkaline phosphatase-positive colony-forming units do not have the capacity for osteoblastic differentiation, in which case we may have overestimated the number of osteoblast progenitor cells in the current study.

The second goal of this study was to determine how the number and concentration of osteoblast progenitor cells obtained with aspiration were influenced by aspiration volume. The data presented here document significant differences between three volumes, and these differences are primarily due to dilution with peripheral blood. These data have important clinical implications and can be used to provide a basis for recommendations regarding aspiration techniques for obtaining human bone-marrow for clinical purposes. Additional studies will be necessary to determine how the prevalence, number, and function of connective-tissue progenitor cells vary as a function of age, gender, menopausal status, pharmaceutical intervention, and selected diseases.

In summary, our data indicate that no one volume is optimum for the achievement of all of the possible goals of aspiration. The intent of the aspiration should dictate the volume that is selected. In the setting of clinical bone-grafting, the volume of the bone graft to be used often is defined by the local anatomy. Therefore, the number of alkaline phosphatase-positive colony-forming units that are placed in a graft is determined by the concentration of such units in the original bone-marrow aspirate. These data indicate that, in the setting of bone-grafting, the aspiration volume should be limited to two milliliters or less in order to maximize the number of alkaline phosphatase-positive colony-forming units in the graft site. We estimate that four one-milliliter aspirates will provide almost twice the number of alkaline phosphatase-positive colony-forming units as one four-milliliter aspirate. Our recommendation is based on the assumption that increasing the number of alkaline phosphatase-positive colony-forming units in a bone-graft site will improve the performance of the graft. Although this hypothesis has not been tested in vivo, the data that Connolly et al.15 obtained with use of implanted diffusion chambers are strongly supportive.

However, if the primary goal is to obtain as many bone-marrow-derived cells or alkaline phosphatase-positive colony-forming units as possible with the minimum number of aspirates, and if the concentration of alkaline phosphatase-positive colony-forming units is not critical, then an aspiration volume of four milliliters is preferable. We estimate that a four-milliliter aspirate will yield 85 per cent or more of the aspiratable bone-marrow cells at a given site. A four-milliliter aspirate may be appropriate for obtaining bone marrow for a bone-marrow-transplantation procedure, as the success of the transplantation depends primarily on the total number of bone-marrow cells rather than on the concentration of cells in the initial sample. A similar recommendation would apply to bone-marrow-derived bone graft if intraoperative processing of the bone marrow was used to concentrate alkaline phosphatase-positive colony-forming units or nucleated cells, by exclusion of the red blood cells and the plasma, for example. However, our data suggest that, even in the setting of bone-marrow transplantation, obtaining more than four milliliters of bone marrow from each aspiration site is probably contraindicated. Larger-volume aspirates contribute little to the over-all number of bone-marrow cells and result principally in unnecessary blood loss.


   Footnotes
 

*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were The Cleveland Clinic Foundation, The Julio Crespo Fund, The Elena Janulis Fund, and Grant R01 AR42997 from the National Institutes of Health.

{dagger}Departments of Orthopaedic Surgery (G.F.M.), Biomedical Engineering (C.B.), and Biostatistics (K.E.), The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195. E-mail address for Dr. Muschler:muschler@bme.ri.ccf.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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