Abstract
Background: Bone marrow aspirated from the iliac crest contains
progenitor cells that can be used to obtain bone-healing of nonunions.
However, there is little available information regarding the number and
concentration of these cells that are necessary to obtain bone repair. The
purpose of this study was to evaluate the number and concentration of
progenitor cells that were transplanted for the treatment of nonunion, the
callus volume obtained after the transplantation, and the clinical healing
rate.
Methods: Marrow was aspirated from both anterior iliac crests,
concentrated on a cell separator, and then injected into sixty noninfected
atrophic nonunions of the tibia. Each nonunion received a relatively constant
volume of 20 cm3 of concentrated bone marrow. The number of
progenitor cells that was transplanted was estimated by counting the
fibroblast colony-forming units. The volume of mineralized bone formation was
determined by comparing preoperative computerized tomography scans with scans
performed four months following the injection.
Results: The aspirates contained an average (and standard deviation)
of 612 ± 134 progenitors/cm3 (range, 12 to 1224
progenitors/cm3) before concentration and an average of 2579
± 1121 progenitors/cm3 (range, 60 to 6120
progenitors/cm3) after concentration. An average total of 51
× 103 fibroblast colony-forming units was injected into each
nonunion. Bone union was obtained in fifty-three patients, and the bone marrow
that had been injected into the nonunions of those patients contained >1500
progenitors/cm3 and an average total of 54,962 ± 17,431
progenitors. The concentration (634 ± 187 progenitors/cm3)
and the total number (19,324 ± 6843) of progenitors injected into the
nonunion sites of the seven patients in whom bone union was not obtained were
both significantly lower (p = 0.001 and p < 0.01, respectively) than those
in the patients who obtained bone union. The volume of the mineralized callus
measured at four months on the computerized tomography scans of the patients
who had union ranged from 0.8 to 5.3 cm3 (mean, 3.1
cm3). There was a positive correlation between the volume of
mineralized callus at four months and the number (p = 0.04) and concentration
(p = 0.01) of fibroblast colony-forming units in the graft. There was a
negative correlation between the time needed to obtain union and the
concentration of fibroblast colony-forming units in the graft (p = 0.04).
Conclusions: Percutaneous autologous bone-marrow grafting is an
effective and safe method for the treatment of an atrophic tibial diaphyseal
nonunion. However, its efficacy appears to be related to the number of
progenitors in the graft, and the number of progenitors available in bone
marrow aspirated from the iliac crest appears to be less than optimal in the
absence of concentration.
Level of Evidence: Therapeutic Level III. See
Instructions to Authors for a complete description of levels of evidence.
The osteogenic capacity of bone marrow was first demonstrated in
rabbits as early as 1869 by
Goujon1. Since the
1960s, some
authors2-4
have shown that osteogenic stem cells in bone marrow are responsible for the
biological efficacy of cancellous bone. This capacity has already been
exploited, by several
investigators5,6,
to reinforce the osteogenic properties of bone allograft by mixing the graft
with bone marrow removed during surgery. In animal experiments, Connolly et
al.7 demonstrated a
positive correlation between bone-marrow osteogenic capacity and cell
concentration, and nonunions have been treated successfully clinically with
autologous bone-marrow grafting
alone8,9.
However, the authors of the clinical studies did not report the number of
connective-tissue progenitor cells that were transplanted, and we are not
aware of any study indicating the number of progenitor cells required to
obtain bone-healing in the treatment of nonunions in humans. Furthermore, only
limited clinical experience with the use of intraoperative centrifugation of
marrow for bone-grafting has been reported.
The purpose of the present study was to evaluate the number and
concentration of progenitor cells that were transplanted for the treatment of
fracture nonunions, the callus volume after transplantation of the
concentrated bone marrow, and the rate of clinical union.
Operative Technique
Marrow Aspiration
Marrow was aspirated from the two anterior iliac crests with the
patient under general anesthesia. After deep insertion of a beveled needle (6
to 8 cm in length and 1.5 mm in internal diameter) into spongy bone, the
marrow was aspirated into a 10-mL plastic syringe. At a given depth, the
needle was turned 45° to reorient the bevel during successive aspirations,
so that the largest possible space was aspirated. After one full turn, the
needle was moved 1 cm toward the surface through the same insertion site, and
aspirations were again performed, with the needle always turned 45° after
each aspiration. The marrow was aspirated in small fractions (4 mL) to reduce
the degree of dilution by peripheral
blood10. Three,
four, or five perforations were made, through the same skin opening, into the
iliac crest, with the perforations spaced approximately 2 cm from each other
to avoid dilution by aspiration in the previous hole. All aspirates were
pooled in plastic bags containing an anticoagulant solution (citric acid,
sodium citrate, and dextrose). Pooled aspirates were then filtered to separate
cellular aggregates and fat (Hemoset NSR LP; B-Braun, Bethlehem,
Pennsylvania).
Concentration
Concentrated buffy coat was obtained after a five-minute centrifugation at
1200 g on a cell separator (Cobe 2991; Gambro BCT, Lakewood, Colorado). This
centrifugation forces the polynuclear cell layer, which is heavier because of
the volume of its nuclei, to the periphery, where it can be collected and
separated from the remainder. The lighter layer with anuclear red cells is
found in the center and is also removed. The buffy coat contains progenitor
cells but also other mononuclear cells, and some of these other cells may be a
source of angiogenic or osteogenic
cytokines11 with a
clinical effect. This centrifugation method reduces a 300-mL bone-marrow
aspirate to a concentrated buffy coat of about 50 mL, which is poured into a
syringe for injection.
Intraosseous Reinjection
A trocar identical to that used to aspirate the marrow was placed both in
the nonunion gap and around the bone ends. The tip of the trocar was
positioned with use of an image intensifier
(Fig. 1). The fibrous tissue of
the nonunion site was not removed or disturbed. The marrow was injected slowly
at a rate of about 20 mL/min. After injection, the trocar, with the stylet in
place, was gradually withdrawn with small oscillating motions (backward and
forward) to fill in the path of the trocar.
Patient Demographics
Sixty patients with an established nonunion of the tibial shaft were
treated with this technique at the same center between 1990 and 2000.
Thirty-eight patients were male. Twenty-eight patients had comorbidities:
fifteen had a history of tobacco use, eight had a history of alcohol abuse,
three had diabetes, and two had used a pharmaceutical agent affecting bone
marrow physiology. Seventeen fractures were in the proximal part of the tibia,
twelve were in the distal part of the tibia, and thirty-one were in the
midpart of the tibial shaft. The patients ranged in age from eighteen to
seventy-eight years, with a mean age of forty years. There were twelve
isolated, closed, low-energy fractures of the tibial shaft, which had been
treated nonoperatively with a plaster-of-Paris cast. There were forty-eight
open fractures, which had been treated with external fixation (a monolateral
frame was used for four cases and a biplanar frame, for forty-four). The
majority (fifty-seven) of the patients underwent definitive fracture fixation
with external fixation or a cast immediately (within twenty-four hours) after
the injury. Three patients underwent changes in the external fixation during
the first week. All patients with an open fracture received routine
soft-tissue management, including débridement, irrigation, and
definitive wound closure, immediately or within three days. According to the
Gustilo-Anderson classification of open
injuries12,
thirty-six fractures were type I, eight were type II, two were type IIIA, one
was type IIIB, and one was type IIIC. The type-IIIB open fracture required
flap coverage, and the type-IIIC open fracture required a vascular repair. Of
the forty-eight open fractures, seven (five type I, one type IIIA, and one
type IIIC) had already been treated, in another center, with an autologous
cancellous bone graft between the fourth and seventh month after the injury.
The graft was harvested with an open procedure from the anterior iliac crest
in five patients and from the posterior iliac crest in two at an average of
four months (range, three to six months) before the aspiration of the bone
marrow graft; the aspiration was done at a minimum of 2 cm from the area of
the previous graft harvest.
The definition of nonunion was a failure of the fracture to heal in six
months in a patient in whom progressive repair had not been observed
radiographically between the third and sixth month after the fracture. All
nonunions were considered atrophic because they showed very little callus
formation. The time between the fracture and the bone marrow injection ranged
from six to twelve months (average, eight months and three weeks).
At the time of the bone marrow grafting, the nonunions were considered to
be not infected according to preoperative assessment for the presence of
systemic infectious parameters. The absence of infection was confirmed
postoperatively by the results of culture of aspirate obtained from the
nonunion site just before the bone marrow injection.
The displacement of the bone fragments at the time of the bone marrow
grafting was measured, on anteroposterior and lateral radiographs, as a
percentage of the width of the bone at the level of the fracture. Fragment
displacement ranged from 0% to 20%, with an average of 6%. The maximum gap
between the fragments was always <5 mm.
Management of the Nonunions Before and After Bone Marrow
Grafting
Anteroposterior and lateral radiographs and a computerized tomography scan
were made preoperatively. Postoperatively, radiographs were made at three and
four weeks to assess the appearance of the callus and then every month
thereafter, until bone healing occurred, to monitor the progression of the
callus.
The volume of mineralized callus was calculated from measurements made on
the computerized tomography scans. The area of callus resulting from the
injection was considered to be the sector of new bone formation between the
time of the preoperative computerized tomography and the time of the
computerized tomography performed four months postoperatively. The protocol
for the computerized tomography scanning consisted of 3-mm-thick sections over
a length of 6 cm at the level of the nonunion (3 cm proximal and 3 cm distal).
The level of each cut was controlled visually to be certain that the
measurements on the preoperative and follow-up scans were made at the same
level. The images were analyzed with use of a set of custom algorithms to
determine pixel intensity and the extent of mineralized bone surface at the
fracture site. Because unmineralized tissue cannot be evaluated accurately
with computerized tomography, it was not analyzed in this study. Although the
computerized tomography scan is two-dimensional, it provides information from
a three-dimensional slab. For each slice, the volume of callus was calculated
by multiplying the area of callus by the slice thickness. The total volume of
callus was the sum of the individual volumes of each slice. Computerized
tomography was not used to determine union or as a guide to ascertain when to
allow full weight-bearing.
The only therapeutic intervention performed in the present study was
percutaneous injection of bone marrow. The same external fixation (for the
open fractures) or plaster-cast immobilization (for the closed fractures) was
used after the bone marrow grafting. All of the patients were treated with a
standard protocol during the first month following the injection. As they had
atrophic nonunion and mobility at the fracture site, weight-bearing was not
allowed during the first month following the injection to avoid mechanical
disruption of the tissue-regeneration and bone-healing processes. After one
month, if (and only if) callus was observed on radiographs, partial
weight-bearing was allowed with the plaster cast or external fixation in
place. There was a one-month transition period between the beginning of
partial weight-bearing and that of full weight-bearing. At the end of that
month, if the patient had no pain and there was cortical bridging or
disappearance of the fracture lines on at least three of the four cortices
viewed on the anteroposterior and lateral radiographs, the plaster cast or the
external fixation was removed.
The treatment was considered to be a success when there was definite
radiographic evidence of fracture union and fulfillment of the clinical
criteria of healing within six months after the autologous bone-marrow
grafting. The clinical criteria of healing included full weight-bearing and no
tenderness at the fracture site on palpation. When a patient did not have
bone-healing six months after the bone marrow grafting, a secondary
intervention to promote fracture union was proposed to him or her and the
treatment was considered a failure. Each patient was followed for at least
three years after the bone marrow grafting.
Bone Marrow Analysis
To measure the number of connective-tissue progenitor cells that were
transplanted, we used the fibroblast colony-forming unit (CFU-F) as an
indicator of stromal cell activity. The fibroblast is not an osteogenic cell
but, according to the theory of pluripotential cell lines, osteocytes develop
from colony-forming-unit progenitor cells in the
marrow11,13-15.
There seems little doubt that these colonies are clonal (i.e., originate from
a single cell), and in this paper the terms "stem cell,"
"connective-tissue progenitor cell," "progenitor," and
"CFU-F" will be considered synonymous. The aggregate of the marrow
was cultured in vitro before and after concentration in order to determine how
much the concentration process altered the number of stem cells in the sample.
The number of nucleated cells was counted with use of a standard Malassez
hemocytometer (Polylabo, Strasbourg, France). Cells were washed once and
resuspended in Hanks balanced salt solution without Ca++ or Mg++. Buffy coats
were collected after centrifugation of the aspirates at 1200 g for ten
minutes.
For the fibroblast colony-forming units (CFU-F), quadruplicate aliquots of
2 × 106 cells were inoculated in 25-mL2
tissue-culture flasks containing 10 mL of culture medium supplemented with 20%
fetal calf serum, 1% L-glutamine, penicillin (100 U/mL), and streptomycin (100
mg/mL). The culture flasks were placed in a humidified incubator with 5%
CO2 and maintained at 37°C. The growth medium was completely
renewed every three to four days, and the cultures were evaluated on the tenth
day. Fibroblast colonies were Giemsa-stained and were counted under an
inverted microscope at 25× magnification. An aggregate of cells
containing more than fifty fibroblasts was scored as a colony. Results were
expressed as the mean number of fibroblast colony-forming units per
106 bone marrow cells. The fibroblastic nature of the colonies was
demonstrated by immunofluorescence staining with antibodies against
fibronectin and type-I and III collagen.
Statistical Methods
Data are reported as the mean and standard deviation, and the significance
level was set at a probability value of <0.05. The outcome variables were
the success of the treatment, the volume of the mineralized callus at four
months, and the time needed to obtain union after the bone marrow grafting.
The therapeutic factors that could influence the outcome variables were the
total number and the concentration of fibroblast colony-forming units injected
at the nonunion site. The patient and fracture variables included age, sex,
associated comorbidities, fracture displacement, and type of open injury
according to the classification of Gustilo and Anderson. A multivariate
analysis was conducted to evaluate the relationship between the outcome and
the set of variables. Correlations between the outcome variables and the cell
factors were determined with use of the Spearman correlation test. The
nonparametric Mann-Whitney U test was used to identify the significance of the
differences between groups. The chi-square test was used to identify trends
within groups with categorical variables.
None of the patients had complications during anesthesia; in
particular, no patient had a decrease in oxygen saturation or a change in
pulse or blood pressure during the procedure. A compartment syndrome did not
develop in any patient after injection of the bone marrow. There were no
infections, hematomas, or chronic pain at the site of the bone marrow
injection.
Patient and Bone Marrow Variables
(Table I)
An average of 306 ± 24 mL of marrow was aspirated from the two iliac
crests of each patient. The number of nucleated cells obtained from the
individual patients ranged from 1 to 24 million/mL, with a mean of 18 ±
7 million/mL. The mean number of fibroblast colony-forming units per one
million nucleated cells obtained from the individual patients ranged from 7 to
51, with a mean of 33 ± 8. The number of nucleated cells was found to
decrease significantly with age (Spearman test, p = 0.03), but, with the
numbers available, no significant difference between men and women was found
(p = 0.26). There was no significant change in the prevalence of progenitor
cells with increasing age (p = 0.12) and, when men and women were analyzed
separately, there was no significant change with age in men (p = 0.28);
however, the prevalence of progenitor cells was observed to decrease
significantly with increasing age in women (p = 0.04).
An average of 1 CFU-F/30 × 103 bone marrow nucleated cells
was obtained in the samples incubated in vitro. The bone marrow obtained by
aspiration from the iliac crests contained an average of 612 ± 134
progenitors/cm3 (range, 12 to 1224 progenitors/cm3).
After concentration, the bone marrow contained an average of 2579 ±
1121 progenitors/cm3 (range, 60 to 6120
progenitors/cm3). A mean of 20 cm3 (range, 17 to 22
cm3) of bone marrow graft was injected into each nonunion site. The
average total number of fibroblast colony-forming units injected into each
nonunion site (i.e., the product of the nucleated cells and the prevalence of
progenitors in the bone marrow graft obtained after concentration) was 51
× 103 (range, 1200 to 122 × 103).
Analysis of the total population demonstrated that age had no significant
effect on the total number of progenitor cells received by each patient (p =
0.08). Also, when men and women were analyzed separately, age was found to
have no significant effect on the number of cells received by the men;
however, increasing age was found to be associated with a significant decrease
in the total number of progenitors received by the women (p = 0.04). With the
number of patients available, the comorbidities of smoking, alcohol abuse,
diabetes, and use of pharmaceutical agents were not associated with
significant changes in the population of cells that were harvested.
Outcomes of Management of the Nonunions
Nonunion outcome variables were defined as the success of the treatment;
the healing time; the volume of callus; and the change in displacement,
shortening, or angulation during bone-healing.
Bone union was obtained in fifty-three of the sixty patients, with the
callus typically appearing on radiographs between the third week and the
second month after the injection. Radiographic evidence of fracture union
(Fig. 2) was observed at an
average of twelve weeks (range, four to sixteen weeks). The volume of the
mineralized callus measured at four months on the computerized tomography
scans of these fifty-three patients ranged from 0.8 to 5.3 cm3,
with a mean value of 3.1 cm3. During healing after the bone marrow
grafting, shortening ranged from 0 to 25 mm, with an average of 5.4 mm. Fifty
nonunions healed with <15 mm of shortening, and three healed with >15 mm
of shortening. During healing, forty-nine patients did not have an increase of
3° in angulation in the frontal plane, and fifty patients did not have an
increase of 3° in the sagittal plane. Four patients had an increase in
angulation of between 3° and 7° in the frontal plane, and three
patients had an increase in angulation of between 3° and 8° in the
sagittal plane. Forty-six nonunions healed with no more displacement in any
plane, and seven had an increase in displacement (ranging from 5% to 10%)
during healing.
Of the sixty patients, seven did not have union, with the volume of
mineralized callus in those patients measuring <0.5 cm3 on the
computerized tomography scan. Three of the seven patients had an increase in
angulation of >10° and an increase in displacement of >20%. These
seven patients required additional surgery to achieve healing. Intramedullary
nailing was performed in three; open bone-grafting, in three; and fibular
osteotomy, in one.
Statistical Analysis
Success of treatment: Of the variables that were explored with
multivariate analysis, the number of transplanted cells was deemed to be the
most relevant to the outcome (Figs.
3 and
4). As the volume of the graft
was relatively constant (average, 20 cm3; range, 17 to 22
cm3), the concentration of transplanted cells also appeared to be
relevant to the outcome. The bone marrow grafts used for the fifty-three
patients who subsequently had bone union contained a mean of 2835 ±
1160 progenitors/cm3 and a mean of 54,962 ± 17,431
progenitors in total, and all of the grafts in these patients contained
>1500 progenitors/cm3. The grafts used in the seven patients in
whom the treatment failed contained a significantly lower concentration (mean,
634 ± 187 progenitors/cm3, p = 0.001) and total number
(mean, 19,324 ± 6843, p < 0.01) of progenitor cells compared with
the patients in whom the treatment was successful. All seven patients with
failure of union had been treated with a bone marrow graft that contained
<1000 progenitors/cm3 and <30,000 progenitors in total. With
the numbers available, the age, sex, and comorbidities of the patients did not
significantly affect the success of the treatment (p > 0.05).
Healing time: There was a negative correlation between the time
needed to obtain union and the concentration of fibroblast colony-forming
units in the graft (Rs [Spearman correlation test] = -0.2, p = 0.04). Fracture
type also had a significant relationship with the time to union (p = 0.03),
with type-II and type-III open fractures taking longer to heal (average,
fourteen weeks) than closed fractures and type-I open fractures (average,
eight weeks). In addition, the location of the fracture had a relationship
with the time until healing (p = 0.03), with the distal fractures taking
longer to heal (average, thirteen weeks) than the proximal fractures (average,
nine weeks). Finally, there was a significant relationship between
comorbidities and the time to union (p = 0.04): the twenty-eight patients with
one or more comorbidities had a longer time until healing (average, fourteen
weeks) than the other patients (average, 10.5 weeks).
Volume of callus: There was a positive correlation between the
volume of mineralized callus at four months and the number and concentration
of fibroblast colony-forming units in the graft (correlation coefficient, 0.3
and 0.6; p = 0.04 and 0.01, respectively). With the numbers available, age,
sex, comorbidities, and fracture type had no significant influence on the
volume of the callus (p = 0.61, 0.24, 0.34, and 0.28, respectively).
During the past two decades, numerous techniques have been developed
to treat fracture nonunions, ranging from invasive interventions (including
internal fixation with the use of bone graft or bone graft substitutes) to
noninvasive procedures (ultrasound and pulsed electromagnetic fields). The
percutaneous technique of autologous bone-marrow grafting that we used is a
minimally invasive alternative.
Our study showed that percutaneous autologous bone-marrow grafting is a
safe treatment for uninfected atrophic nonunions of the tibial diaphysis, as
we encountered no local or systemic complications. One theoretical criticism
of this technique is that there is a risk of fat embolism during the injection
of the bone marrow into the nonunion site. However, in our study, the bone
marrow aspirates were filtered to separate the marrow and fat, and none of the
patients had complications during anesthesia.
Fifty-three of the sixty nonunions healed, which confirms the effectiveness
of this technique for the treatment of atrophic
nonunions8,9,16,17.
Historically, resection of the fibrous tissue at the nonunion site combined
with mechanical stabilization has been described as being essential for the
treatment of an atrophic
nonunion18. In this
series, the trocar was not used to remove the intervening callus or fibrous
tissue. The fibrous tissue interposed between the bone ends ossified after the
injection of the bone marrow. It is difficult to explain the exact mechanism
that allows the transformation of fibrous tissue into callus. Bone marrow was
injected both in the nonunion gap and around the bones. It is not possible to
know whether the injected marrow was able to convert the fibrous tissue into
bone or if the interposed tissue was transformed into bone only after the
bridging callus (obtained from the graft around the bone) stopped micromotion
at the nonunion site and allowed union of the gap.
Like all techniques, this new option of bioactive cell stimulation has its
limitations, one of which is that it has not been evaluated in the presence of
internal fixation (plates or intramedullary nails). One potential weakness of
the present study is the absence of a cohort with a placebo treatment such as
injection of saline solution. Also, the cell counts were determined
retrospectively; thus, we cannot determine if the technique should have been
used as the sole treatment method. Percutaneous injection of bone marrow
cannot be used when there is pre-existing angular deformity or shortening,
both of which require direct access to the nonunion site. As the volume of
callus obtained with this technique is limited, the fracture fragment gap size
and displacement should be limited as well.
Another important finding of this study is the relationship between the
volume of the callus and the number of progenitors in the graft. The addition
of bone-marrow-derived cells has been shown to enhance bone-healing in
animals. There are limited data on the number of progenitors that are resident
in bone marrow grafts in
humans19. The
variability in the osteogenic potential from patient to patient is a
limitation of the technique, and little is known about the extent to which
these cells are susceptible to activation for bone repair after they are
implanted. Because we initially had no data on the number of cells necessary
to obtain bone-healing, the volume of the aspirate and volume of the
transplanted graft were similar for all of the patients. In this series, the
number of progenitors was determined retrospectively, and there was variation
among the patients.
Differences among connective-tissue progenitors harvested from various
individuals are beginning to be understood. These differences depend on many
variables, such as age, gender, and local and systemic
disease10,19-22,
and the variability in the osteogenic potential from patient to patient
represents a limitation of the technique. One of the challenges in the
operating room for the surgeon using this technique could be the evaluation of
the number of cells obtained by aspiration. Bone marrow cellularity declines
with age, and there is also a decrease in the prevalence of connective-tissue
progenitors with increasing
age23,24,
even if this was not evident in our small series of patients. However, as can
be observed by examining the data in our
Table I and the information in
other reports21,
age and gender account for only a fraction of the variation; thus,
connective-tissue progenitors can be obtained by bone marrow aspiration from
patients of all ages. It may be useful for surgeons to know the cellularity of
the bone marrow when operating on older patients. The number of progenitors
can be determined only with a culture, but the quantity of medullary nuclear
cells can be evaluated in the operating room (if necessary) by the equation
presented in the Appendix. Since the total number of progenitors represents
the product of the nucleated cells and the prevalence of progenitors in the
aspirate, a decline in the number of nucleated cells can be corrected by an
increase in the volume of aspiration. However, a larger volume of aspiration
decreases the concentration of progenitor cells because of dilution with
peripheral blood.
Still another important observation in this study was the influence that
the concentration of bone marrow by centrifugation had on the results. The
seven patients who did not obtain union had all received a marrow graft with
<1000 progenitors/cm3 and <30,000 progenitors in total; both
the mean concentration and the mean number were significantly lower than those
for the patients for whom the treatment did not fail. Therefore, it seems
reasonable to suggest that a graft needs to contain >1000
progenitors/cm3. This finding has implications regarding the
intraoperative processing of bone marrow to select progenitors because bone
marrow obtained by aspiration and not concentrated contains only a mean of
approximately 600 progenitors/cm3 (range, 12 to 1224
progenitors/cm3).
Our results confirm that it is important to increase the number of
progenitors in the graft after aspiration. Connolly et
al.7 examined the
possibility of improving the efficacy of an aspirated bone-marrow graft by
concentration in a study of animals. Even if the issue of concentration was
not directly addressed by our experimental design, we were able to confirm its
influence in our clinical study of humans by determining the number of cells
in a standardized volume. However, it is not possible for us to know, from the
findings in this study, whether the same number of cells in a smaller (or
larger) volume would be similarly effective. The importance of the
concentration of cells that can be delivered may be related to the survival of
these progenitors after
transplantation20.
The amount of available oxygen is probably one of the limiting factors after
transplantation. Since the transplanted progenitor cells compete with other
cells for oxygen, one way to optimize cell survival is to limit the
transplanted cells to those that contribute to the formation of bone (i.e.,
exclude all others). This was achieved by centrifugation in our series. Use of
a porous implantable material has been reported as an alternative method for
concentration and selection of connective-tissue
progenitors25.
Other methods to increase the population of progenitors in the bone marrow
graft, such as the use of growth
factors26-29,
will probably be proposed in the future.
The quantity of medullary nuclear cells per kilogram of marrow was
calculated with use of a formula that takes into account blood dilution. It
was estimated that, in each milliliter of aspirate, medullary cells were
represented by the difference between the nuclear cell count and the count in
peripheral blood (sampled during the period of general anesthesia):
N(108/kg)=(V×NP)-(V-100)×NSP
where V = the total volume of aspirate in milliliters, including the
harvesting medium; NP = the nuclear cell count per milliliter in the
collection bag, in which the harvesting medium is included, that leaves the
operating room; V - 100 = the exact volume of aspirate, after subtraction of
the 100 mL of harvesting medium; NS = the nuclear cell count per milliliter of
peripheral blood drawn during the period of general anesthesia; and P = the
patient's weight in kilograms. As an example: for a total final volume of 300
mL containing 14 × 106 nuclear cells/mL, obtained from a
70-kg adult with a leukocyte count of 4 × 106/mL as
determined while the patient is under general anesthesia, it can be estimated
that the medullary nuclear cell count is 5 × 107/kg, for a
total of 0.35 × 1010 nuclear cells. ?
The authors did not receive grants or outside funding in support of their
research or preparation of this manuscript. They did not receive payments or
other benefits or a commitment or agreement to provide such benefits from a
commercial entity. No commercial entity paid or directed, or agreed to pay or
direct, any benefits to any research fund, foundation, educational
institution, or other charitable or nonprofit organization with which the
authors are affiliated or associated.
Goujon E. Recherches
expérimentales sur les propriétés physiologiques de la
moelle des os. J Anat Physiol.1869;6:
399-412.6399
1869
Burwell RG. Studies in the
transplantation of bone. VII. The fresh composite homograft-autograft of
cancellous bone; an analysis of factors leading to osteogenesis in marrow
transplants and in marrow-containing bone grafts. J Bone Joint Surg
Br.1964;46:
110-40.46110
1964
[PubMed]
Beresford JN. Osteogenic stem cells and
the stromal system of bone and marrow. Clin Orthop Relat Res.1989;240:
270-80.240270
1989
[PubMed]
Burwell RG. The function of bone marrow
in the incorporation of a bone graft. Clin Orthop Relat Res.1985;200:
125-41.200125
1985
[PubMed]
Burwell RG. Studies in the
transplantation of bone. 8. Treated composite homograft-autografts of
cancellous bone: an analysis of inductive mechanisms in bone transplantation.
J Bone Joint Surg Br.1966;48:
532-66.48532
1966
[PubMed]
Boehm CA, Muschler GF. Rapid
concentration of bone marrow derived osteoblastic progenitor cells (CFU-Os) in
allograft bone powder. J Bone Miner Res.1999;14(Suppl D):
S474.14S474
1999
Connolly J, Guse R, Lippiello L, Dehne
R. Development of an osteogenic bone-marrow preparation. J Bone Joint
Surg Am.1989;71:
684-91.71684
1989
Connolly JF, Guse R, Tiedeman J, Dehne
R. Autologous marrow injection as a substitute for operative grafting of
tibial nonunions. Clin Orthop Relat Res.1991;266:
259-70.266259
1991
[PubMed]
Healey JH, Zimmerman PA, McDonnell JM,
Lane JM. Percutaneous bone marrow grafting of delayed union and nonunion in
cancer patients. Clin Orthop Relat Res.1990;256:
280-5.256280
1990
[PubMed]
Muschler GF, Boehm C, Easley K.
Aspiration to obtain osteoblast progenitor cells from human bone marrow: the
influence of aspiration volume. J Bone Joint Surg Am. 1997;79:1699-709.
Erratum in: J Bone Joint Surg Am.1998;80:
302.80302
1998
[CrossRef]
Tateishi-Yuyama E, Matsubara H, Murohara
T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, Akashi H,
Shimada K, Iwasaka T, Imaizumi T; Therapeutic Angiogenesis Using Cell
Transplantation (TACT) Study Investigators. Therapeutic angiogenesis for
patients with limb ischaemia by autologous transplantation of bone-marrow
cells: a pilot study and a randomised controlled trial. Lancet.2002;360:
427-35.360427
2002
[PubMed][CrossRef]
Gustilo RB, Anderson JT. Prevention of
infection in the treatment of one thousand and twenty-five open fractures of
long bones: retrospective and prospective analyses. J Bone Joint Surg
Am.1976;58:
453-8.58453
1976
Friedenstein AJ, Petrakova KV,
Kurolesova AI, Frolova GP. Heterotopic of bone marrow. Analysis of precursor
cells for osteogenic and hematopoietic tissues.
Transplantation.1968;6:
230-47.6230
1968
[PubMed][CrossRef]
Owen M. Lineage of osteogenic cells and
their relationship to the stromal cell system. In: Peck WA, editor.
Bone and mineral research. Volume 3.
Amsterdam: Elsevier Science; 1985. p
1-25.31
1985
Owen M, Friedenstein AJ. Stromal stem
cells: marrow-derived osteogenic precursors. Ciba Found Symp.1988;136:
42-60.13642
1988
[PubMed]
Paley D, Young MC, Wiley AM, Fornasier
VL, Jackson RW. Percutaneous bone marrow grafting of fractures and bony
defects. An experimental study in rabbits. Clin Orthop Relat
Res.1986;208:
300-12.208300
1986
Garg NK, Gaur S, Sharma S. Percutaneous
autogenous bone marrow grafting in 20 cases of ununited fracture. Acta
Orthop Scand.1993;64:
671-2.64671
1993
[CrossRef]
Johnson KD. Management of malunion and
nonunion of the tibia. Orthop Clin North Am.1987;18:
157-71.18157
1987
[PubMed]
Hernigou P, Beaujean F. Treatment of
osteonecrosis with autologous bone marrow grafting. Clin Orthop Relat
Res.2002;405:
14-23.40514
2002
[CrossRef]
Hernigou P, Beaujean F. Progeniteurs
ostéoblastiques de la moelle osseuse des crêtes iliaques: leur
variation en fonction de la pathologie, de l'âge, du sexe. Revue
du Rhumatisme.2003;70:
949-50.70949
2003
Muschler GF, Nitto H, Boehm CA, Easley
KA. Age- and gender-related changes in the cellularity of human bone marrow
and the prevalence of osteoblastic progenitors. J Orthop Res.2001;19:
117-25.19117
2001
[PubMed][CrossRef]
Hernigou P, Beaujean F. Abnormalities in
the bone marrow of the iliac crest in patients who have osteonecrosis
secondary to corticosteroid therapy or alcohol abuse. J Bone Joint Surg
Am.1997;79:
1047-53.791047
1997
Quarto R, Thomas D, Liang CT. Bone
progenitor cell deficits and the age-associated decline in bone repair
capacity. Calcif Tissue Int.1995;56:
123-9.56123
1995
[PubMed][CrossRef]
D'Ippolito G, Schiller PC, Ricordi C,
Roos BA, Howard GA. Age-related osteogenic potential of mesenchymal stromal
stem cells from human vertebral bone marrow. J Bone Miner Res.1999;14:
1115-22.141115
1999
[PubMed][CrossRef]
Muschler GF, Nitto H, Matsukura Y, Boehm
C, Valdevit A, Kambic H, Davros W, Powell K, Easley K. Spine fusion using cell
matrix composites enriched in bone marrow-derived cells. Clin Orthop
Relat Res.2003;407:
102-18.407102
2003
[CrossRef]
Muschler GF, Midura RJ. Connective
tissue progenitors: practical concepts for clinical applications. Clin
Orthop Relat Res.2002;395:
66-80.39566
2002
[CrossRef]
Lane JM, Yasko AW, Tomin E, Cole BJ,
Waller S, Browne M, Turek T, Gross J. Bone marrow and recombinant human bone
morphogenetic protein-2 in osseous repair. Clin Orthop Relat
Res.1999;361:
216-27.361216
1999
[CrossRef]
Yoon ST, Boden SD. Osteoinductive
molecules in orthopaedics: basic science and preclinical studies. Clin
Orthop Relat Res.2002;395:
33-43.39533
2002
[CrossRef]
Muschler GF, Nakamoto C, Griffith LG.
Engineering principles of clinical cell-based tissue engineering. J
Bone Joint Surg Am.2004;86:
1541-58.861541
2004