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263Oral & Craniofacial Tissue Engineering

Translational Research: The CD34+ Cell Is Crucial

for Large-Volume Bone Regeneration from the

Milieu of Bone Marrow Progenitor Cells in

Craniomandibular Reconstruction

Robert E. Marx, DDS1/David B. Harrell, PhD, OF, FRIPH2

Purpose: This study investigated the role of the bone marrow–derived CD34+ cell in a milieu of

osteoprogenitor cells, bone marrow plasma cell adhesion molecules, recombinant human bone

morphogenetic protein (rhBMP), and a matrix of crushed cancellous allogeneic bone in the clinical

regeneration of functionally useful bone in craniomandibular reconstructions. The history and

current concepts of bone marrow hematopoietic stem cells and mesenchymal stem cells are

reviewed as they relate to bone regeneration in large continuity defects of the mandible.

Materials and Methods: Patients with 6- to 8-cm continuity defects of the mandible with retained

proximal and distal segments were randomized into two groups. Group A received an in situ

tissue-engineered graft containing 54 ± 38 CD34+ cells/mL along with 54 ± 38 CD44+, CD90+,

and CD105+ cells/mL together with rhBMP-2 in an absorbable collagen sponge (1 mg/cm of

defect) and crushed cancellous allogeneic bone. Group B received the same graft, except the

CD34+ cell concentration was 1,012 ± 752 cells/mL. The results were analyzed clinically,

radiographic bone density was measured in Hounsﬁeld units (HU), and specimens were analyzed

histomorphometrically. Results: Forty patients participated (22 men and 12 women; mean age,

57 years). Eight of 20 group A patients (40%) achieved the primary endpoint of mature bone

regeneration, whereas all 20 group B patients (100%) achieved the primary endpoint. CD34+ cell

counts above 200/mL were associated with achievement of the primary endpoint. Bone density

was lower in group A (424 ± 115 HU) than in group B (731 ± 98 HU). Group A bone showed a

mean trabecular bone area of 36% ± 10%, versus 67% ± 13% for group B. Conclusions: The

CD34+ cell functions as a central signaling cell to mesenchymal stem cells and osteoprogenitor

cells in bone regeneration. The mechanism of bone marrow–supported grafts requires a complete

milieu to regenerate large quantities of functionally useful bone. CD34+ cell counts in a

concentration of at least 200/mL in composite grafts are directly correlated to clinically successful

bone regeneration. O

ral

raniOfaC

issue

2012;2:263–271. doi: 10.11607/octe.0059

Key words: bone regeneration, hematopoietic stem cells, mesenchymal stem cells,

recombinant human bone morphogenetic protein

The link between cells of hematopoietic lineage

and marrow osteoprogenitor cells is not new. As

early as 1763, Albrecht von Haller stated that

“the origin of bone is the artery carrying blood and

in it the mineral content.”1 Additionally, the Conhein

hypothesis of 1867 stated that the bloodstream and

consequently the bone marrow was the source of

cells involved in wound regeneration.2 These early

works established the importance of the vascular sys-

tem and bone marrow, along with their associated cell

1 Professor of Surgery and Chief, Division of Oral and

Maxillofacial Surgery, University of Miami Miller School of

Medicine, Miami, Florida, USA.

2 Director of Cellular Science and Education, Harvest Terumo,

Plymouth, Massachusetts, USA.

Correspondence to: Dr Robert E. Marx, University of Miami

Miller School of Medicine, 9380 SW 150th Street, Suite 190,

Miami, FL 33176, USA. Email: rmarx@med.miami.edu

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Marx/Harrell

264 Volume 2, Number 4, 2012

lines, in the activation of resident cells within bone for

the purpose of bone regeneration and resident cells

within soft tissue for wound healing.

The current understanding of the cellular composi-

tion of bone marrow aspirates (BMA) and bone mar-

row aspirate concentrates (BMAC) used in today’s

craniofacial bone regeneration involves both hemato-

poietic stem cells (HSCs) and mesenchymal (stromal)

stem cells (MSCs). However, the term stem cells cre-

ates confusion in and of itself, as the term is often used

to refer to both true stem cells as well as to progenitor

cells.3,4 This confusion is multiplied by the common

belief that an MSC represents a pluripotent stem cell

and is all that is required for tissue regeneration,5 a

belief that has not been validated to date.6,7 However,

it has been demonstrated that HSCs are directly in-

volved in both bone regeneration and soft tissue re-

generation,8–10 theoretically supporting the concept

of a pluripotent stem cell to regenerate the entire tis-

sue complex. On the other hand, two landmark pa-

pers11,12 (published in 2005 and 2011, respectively)

identiﬁed the paracrine cellular communications and

cell contact between osteoblasts and hematopoietic

stem cell/progenitor cells, suggesting that a multi-

cellular mechanism of MSCs in BMA/BMAC is re-

quired for bone regeneration.

Marx and Tursun identiﬁed four important subsets

of stem cells in blood and in BMA (CD34+, CD44+,

CD90+, and CD105+) and found by polymerase

chain reaction and colony-forming unit (CFU) analysis

that their native amounts in the anterior and posterior

ilium were equal and were more than twice that of the

tibial plateau.13 This was followed by a randomized

clinical study, which demonstrated useful and durable

bone regeneration in maxillary alveolar defects of small

volume when combined with platelet-rich plasma,

which contains only small numbers of these MSCs/

progenitor cells, and recombinant human bone mor-

phogenetic protein (rhBMP) (Figs 1 and 2).14 Although

these circulating stem cells/osteoprogenitor cells

proved to be adequate in these smaller defects with

abundant host MSCs/osteoprogenitor cells, they were

found to be incapable of large-volume bone regenera-

tion in continuity defects, where few (if any) host site

resident stem cells or osteoprogenitor cells exist (Fig 3).

Therefore, it became apparent that large continuity

Fig 3 Low numbers of circulating stem cells, even when com-

bined with rhBMP and cancellous allogeneic bone, will not pre-

dictably regenerate bone in large mandibular continuity defects.

Fig 1 Small-volume alveolar ridge bone regeneration was ac-

complished with platelet-rich plasma, rhBMP, and cancellous al-

logeneic bone.

Fig 2 Graft seen in Fig 1; it was capable of receiving dental im-

plants without further augmentation.

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Marx/Harrell

265Oral & Craniofacial Tissue Engineering

defects of the jaws required (1) identiﬁcation of the

crucial cells in bone marrow that control bone regen-

eration and (2) a means of increasing their numbers.

Several studies have implied that bone marrow

CD34+ cells might be the pivotal cells controlling bone

regeneration. The CD34+ cell has been considered an

HSC.9,15–17 However, studies in the early 2000s iden-

tiﬁed it as a cell capable of plasticity18; ie, capable of

differentiation between MSC and HSC lineages.19,20

This concept reinforced the notion that a single cell line

can differentiate into the full cellular tissue composite of

bone, blood vessels, lymphatic tissue, endosteum, peri-

osteum, and bone marrow once again.5 Experiments by

Matsumoto et al conﬁrmed the multilineage differentia-

tion of circulating CD34+ cells, resulting in endothelial

cells for vasculogenesis and osteoblasts for bone regen-

eration.8 This ﬁnding was conﬁrmed by others,3 proving

that clonal cells of HSC isolated from bone marrow pro-

duce CFU-ﬁbroblasts (CFU-F) and ﬁbrocytes that were

not of MSC origin but of HSC origin.21,22

The issue that remains under debate is whether

the bone marrow–derived CD34+ cell controls bone

regeneration via its own multilineage differentiation,

followed by expansion or by paracrine and autocrine

signaling to other bone marrow precursor cells and

even by signaling to its parents or progeny to create a

multilineage differentiation and expansion to regener-

ate bone that then undergoes remodeling and renewal.

To explore this issue, the present study used two

concentrations of bone marrow–aspirated CD34+

cells in a proven in situ tissue engineering model.14 This

model combines the selected CD34+ concentration

and bone marrow osteoprogenitor cells with rhBMP-2/

absorbable collagen sponge (rhBMP-2/ACS) and a

matrix of crushed cancellous allogeneic bone, thus com-

pleting the classic tissue-engineering triangle (Fig 4).

MATERIALS AND METHODS

With approval from the institutional review board, pa-

tients with 6- to 8-cm continuity defects of the mandible

were randomized to receive treatment with one of two

protocols. Group A patients received (1) BMA con-

taining total nucleated cells (TNC) 15.5 ± 10

/mL and

54 ± 38 cells/mL CD34+ CFU-F; (2) rhBMP-2/ACS,

1 mg/cm of defect; (3) crushed cancellous allogeneic

bone (University of Miami Tissue Bank); and (4) BMA

containing CD44+, CD90+, and CD105+ osteopro-

genitor cells 15.5 × 10

TNC and 54 ± 38 cells/mL

CFU-F of each. Group B patients received the same

materials, except that the CD34+ BMA contained

TNC 98 ± 32 × 10

/mL and 1,012 ± 752 cells/mL

CD34+ CFU-F.

Method of Bone Marrow Harvest and

Concentration

A total of 60 mL of autologous bone marrow was

harvested from each of four puncture sites in the

bilateral anterior iliac crest intraoperatively at the

point of care using a heparinized trocar to aspirate

15 mL from each site, per the protocol of Marx and

Stevens.

Each 60-mL marrow harvest was placed

into a blood transfer bag into which 4 mL of anti-

coagulant citrate dextrose-A had been placed. In

group A patients, 10 mL of this anticoagulated

BMA was used as the CD34+ cellular leg and

the CD44+, CD90+, CD105+ cellular legs of the

tissue-engineering triangle, with TNC counts and

CFU-F cell counts as noted previously. In group

B patients, 60 mL of BMA was processed using a

Smart Prep2 BMAC system (Harvest), which uses

a gradient density centrifugation to provide 10 mL

of CD34+ rich BMAC with the TNC and CFU-F cell

counts as noted earlier.

Patients

Inclusion criteria for the study were:

1. Age over 18 years

2. Radiographic evidence of healed proximal and

distal bone segments in good alignment

3. Sufﬁcient soft tissue to cover a graft without the

need for a local or distant ﬂap

Exclusion criteria were as follows:

1. Previous procedure entering bone marrow

2. History or presence of bone marrow pathology

3. Hypersensitivity to heparin, BMP, or anticoagulant

citrate dextrose-A

4. Previous radiation to the jaws

5. Metastatic cancer

Fig 4 The classic tissue-engineering triangle for predictable tis-

sue regeneration: cells, signal, and matrix.

Matrix

Cells Signal

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Marx/Harrell

266 Volume 2, Number 4, 2012

6. Exposure to an oral bisphosphonate less than

9 months previous

7. Current regimen or history of receiving an intra-

venous bisphosphonate, methotrexate, or pred ni-

sone

Surgical Procedure

Each patient’s continuity defect was exposed through

a submandibular incision. The recipient tissue bed

was developed by removing scar tissue and reﬂecting

a minimum of 4 cm of periosteum from both the proxi-

mal and distal segments and scoring the surface edge

of each bone segment to remove the cortex.

The graft was prepared by solubilizing the rhBMP-2

for 5 minutes according to the manufacturer’s direc-

tions (Infuse Bone Graft, Medtronics) and applying it

to the ACS. The rhBMP-2 was allowed a minimum of

15 minutes to bind to the ACS to achieve 95% binding.

Cubes of crushed cancellous allogeneic bone (Uni-

versity of Miami Tissue Bank) were passed through

a bone mill (Stryker Corp) one time using 10 mL

of premilled bone per centimeter of mandibular defect.

The respective anticoagulated BMA or bone marrow

concentrate was added to the milled crushed cancel-

lous allogeneic bone and mixed thoroughly. Then the

saturated rhBMP-2/ACS was cut into 1-cm square

pieces and added to the mixture to create a compos-

ite graft. Prior to placement, 0.5 mL of a 10% cal-

cium chloride solution containing 5,000 IU of bovine

thrombin was added to the composite graft to reverse

the anticoagulant, release the growth factors inherent

in the platelet fraction, and activate the cell adhesion

molecules in the plasma fraction.

This composite graft (Fig 5) was placed and con-

densed into the prepared continuity defect using Pen-

ﬁeld bone packers (Fig 6). The graft was contained

with either a titanium mesh or an allogeneic bone strut,

as appropriate to the morphology of the defect. Four

weeks of maxillomandibular ﬁxation followed for each

patient, along with 1 week of postoperative antibiotics

and appropriate analgesics.

Assessments and Follow-up

All patients underwent examination and release of

maxillomandibular ﬁxation and arch bar removal under

local anesthesia at 4 weeks. A baseline cone beam

computed tomographic (CT) scan was taken at that

time. Examinations and cone beam CT scans were

also performed at 3 and 6 months. At 6 months, the

principal investigator determined whether there was

sufﬁcient bone and sufﬁcient mineral density to ac-

commodate dental implants (primary endpoint). Those

patients in whom the clinical and radiographic assess-

ments indicated implant placement received implants

(Biomet 3i), and a core or open bone biopsy specimen

was harvested. All implants were allowed 6 months for

osseointegration before functional loading.

Study Endpoints

The primary endpoint was sufﬁcient bone to place dental

implants without the need for additional grafting. Patients

who regenerated sufﬁciently dense bone capable of im-

plant primary stability were considered responders. One

secondary endpoint was the radiographic density of the

bone in Hounsﬁeld units (HU) taken from the 6-month

cone beam CT scan. Another secondary endpoint was

the analysis of trabecular bone density obtained from

histomorphometric analysis of the bone cores or biopsy

specimens taken at implant placement using the Image-

Pro Plus 5.0 computer-based analyzer.

Statistical Analysis

Data evaluation was accomplished using the sta-

tistical software package SPSS 13.0 (SPSS Inc).

Discrete variables were presented as cell counts and

percentages. A paired t test was used to analyze the

Fig 5 Composite bone graft of BMAC (CD34+ cell–enriched),

rhBMP, and cancellous allogeneic bone.

Fig 6 At surgery, the composite graft

shows a loose consistency.

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Marx/Harrell

267Oral & Craniofacial Tissue Engineering

BMA and BMAC cell counts. A multivariate logistic re-

gression analysis was used to study predictors of clin-

ical beneﬁt after BMA and BMAC graft applications.

For all analyses, P < .05 was considered signiﬁcant.

RESULTS

Forty patients (mean age, 57 years; range, 19 to 78

years; 22 men, 12 women) participated in the study.

All patients proceeded through the postoperative

course without signiﬁcant complications and showed

evidence of new bone regeneration by 6 months.

Table 1 identiﬁes achievement of the primary end-

point in each group. Eight of 20 group A patients

(40%) achieved the primary endpoint (Figs 7 and 8),

whereas all 20 group B patients (100%) achieved the

primary endpoint (Figs 9 and 10); this difference was

statistically signiﬁcant (P = .006). This correlates to a

CD34+ cell count of 54 ± 38 cells/mL in group A ver-

sus a CD34+, cell count of 1,012 ± 752 cells/mL in

group B, while the concentrations of progenitor cells

of CD44+, CD90+ and CD 105+ were nearly equal

between the two groups.

According to univariate analysis, a CD34+ cell count

of 200/mL CFU-F was associated with a clinical out-

come of sufﬁcient bone regeneration to accommodate

Fig 7 Group A graft that did not achieve the primary endpoint. Bone height, quan-

tity, and maturity were inadequate at 6 months.

Fig 8 Group A graft cone beam CT slice in-

dicating bone regeneration with voids and the

absence of a cortical rim.

Fig 9 Group B graft that met the primary endpoint. Bone height, quan-

tity, and maturity are present at 6 months.

Fig 10 Group B graft cone beam

CT slice indicating complete bone

regeneration with a cortical rim and

trabecular bone without voids.

Table 1 Results of Treatment

Endpoint

Group A

(n = 20)

Group B

(n = 20) P

Regeneration of

implantable bone

8/20 (40%) 20/20 (100%) .006

Mean radiographic

density

424 ± 115 HU 731 ± 98 HU .01

Mean trabecular

bone area

36% ± 10% 67% ± 13% .01

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Marx/Harrell

268 Volume 2, Number 4, 2012

dental implant placement and gain primary stability

without the need to augment the regenerated bone

(P = .011, odds ratio 5.1, 95% conﬁdence interval

1.25 to 20.52) (Fig 11).

Table 1 also illustrates the mean radiographic den-

sity of the regenerated bone in both groups. Group

A sites showed only 58% of the density of group B

sites (424 ± 115 HU and 731 ± 98 HU, respectively)

(Figs 12 and 13) (P = .01). The trabecular bone

area of group A (36% ± 10%) was only 54% of the

group B trabecular bone area (67% ± 13%) (Figs 14

to 17) (P = .01).

Fig 14 Group A graft with trabecular bone area of 28%. Note the

thin trabecular bone struts and the ﬁbrous-fatty marrow spaces that

occupy much of the volume instead of bone (hematoxylin-eosin;

original magniﬁcation ×1.6).

Fig 16 Group B graft with trabecular bone area of 74%. Note

the thick bone trabecular struts and regeneration of marrow cells

rather than ﬁbrous-fatty marrow (hematoxylin-eosin; original mag-

niﬁcation ×1.6).

Fig 15 Group A graft at higher power. Mature bone with lamellar

architecture but insufﬁcient quantity and thin trabecular struts is

present; haversian canals are absent (hematoxylin-eosin; original

magniﬁcation ×10).

Fig 17 Group B graft at higher power. Mature bone shows la-

mellar architecture, small cellular marrow spaces, and haversian

canals (hematoxylin-eosin; original magniﬁcation ×10).

Fig 12 Radiographic bone

density of a group A graft

(512 HU).

Fig 13 Radiographic bone

density of a group B graft

(794 HU).

Fig 11 Complete bone regeneration occurs if the CD34+ cell

count exceeds 200/mL. Here, a CD34+ cell count of 292/mL

provided the required crucial cell count in the tissue-engineering

triangle.

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Marx/Harrell

269Oral & Craniofacial Tissue Engineering

DISCUSSION

This study investigated the role of the bone marrow–

derived CD34+ cell in a milieu of osteoprogenitor

cells, bone marrow plasma cell adhesion molecules,

rhBMP, and a matrix of crushed cancellous allogeneic

bone in the clinical regeneration of functionally useful

bone in craniomandibular reconstructions. The main

ﬁndings can be summarized as follows:

1. The CD34+ cell as an HSC is the crucial cell

among the other stem cells/osteoprogenitor cells

in regenerating bone in an in situ human tissue-

engineering model.

2. A CD34+ cell count of 200/mL CFU-F or greater is

directly correlated to a successful clinical outcome.

3. The correlation of extensive bone regeneration

with higher counts of CD34+ cells as compared

to lower counts of CD34+ cells together with

equal counts of other MSC/osteoprogenitor cells

and cell adhesion molecules in the milieu strong-

ly supports the mechanism of action proposed

by others; that is, a master signaling cell using

paracrine and autocrine cellular cross talk to up-

regulate other bone marrow stem cells/progenitor

cells of either HSC or MSC origin, resulting in sig-

niﬁcant bone regeneration within the environment

of the graft.12,24–26

CLINICAL IMPLICATIONS

This study demonstrates that BMA by itself is in-

sufﬁcient to predictably regenerate clinically useful

bone in large bony continuity defects of the man-

dible. Concentrations of bone marrow to achieve a

CD34+ cell count of at least 200 CFU-F/mL are

necessary to provide predictable bone regeneration

in the context of the composite graft system used in

this study. Therefore, devices must be capable of con-

centrating anticoagulated bone marrow to ﬁve to eight

times baseline levels and to particularly focus on the

CD34+ cell population while still retaining baseline

levels of CD44+, CD90+, and CD105+ cells.27

Improved Understanding of Adult

Bone Marrow Stem Cells

Beyond using the results of this stem cell study to im-

prove clinical outcomes, this study also sheds some

light on the understanding of cell-to-cell, cell-to-

matrix, and cell-to-microenvironment interactions

within human adult bone marrow that may result in fur-

ther improvements in clinical outcomes.

Although hematopoeisis was once thought to be the

sole function of bone marrow,28 work by Friedenstein

et al in 1966 identiﬁed bone marrow MSCs other than

HSCs, thereby deﬁning two distinct classes of multi-

potent human bone marrow stem cells: HSCs and

MSCs.29 They isolated CFU-F multipotent cells from

marrow plastic adherent cells and found that they could

differentiate into osteoblasts, chondrocytes, adipocytes,

and supporting stroma for hematopoietic cells. Hence,

they have often been referred to as mesenchymal stro-

mal cells as well as mesenchymal stem cells.

HSCs were originally thought to solely produce the

lineage of red and white blood cells and thrombocytes/

platelets and reconstitute the bone marrow stem cell

population. However, more recent studies have identi-

ﬁed that HSCs are signiﬁcantly plastic, producing and

up-regulating MSCs and their progeny, and are not

limited to blood cell production.20,30,31 Researchers

now realize that these two classes of stem cells are

not independent of each other but are actually very in-

terdependent.31,32 Furthermore, each is inﬂuenced by

its own anatomical niche,10,11,33 various growth fac-

tors,25,34 and by the local microenvironment into which

it may be placed.35

In the context of this study, the HSCs were rep-

resented by the CD34+ cell and the MSCs by the

CD44+, CD90+, and CD105+ cells. Each cell

responded to the peripheral signals in the micro-

environment, represented by the rhBMP-2 as well as

the inherent hypoxia in the wound, the growth factors

from the platelets and macrophages, and the cell ad-

hesion molecules present throughout the graft. The

subsequent cellular proliferations were directed by

the CD34+ cell, and the osteoblast differentiation

and osteoid synthesis were directed, up-regulated,

and stimulated by all of the cellular components,

growth factors, and matrix proteins in the graft. These

cell-to-cell, cell-to-matrix, and growth-factor-to-cell

interactions resulted in the useful bone regeneration

observed and emphasize the importance of cellular

heterogeneity in graft systems.

Although signiﬁcant research efforts have been

made to identify one speciﬁc cell type or one speciﬁc

growth factor that would signiﬁcantly increase tissue

regeneration, this study and others demonstrate that

a heterogenous population of HSCs and MSCs, de-

livered with numerous but speciﬁc growth factors in

a conducive microenvironment, provides more pre-

dictable bone regeneration. The importance and sup-

portive actions of the various cellular components in

human bone marrow are outlined in Table 2, and the

supportive actions of growth factors and the microen-

vironment are outlined in Table 3. Although the CD34+

cell is a crucial and perhaps outcome-determining

cell, it does not act alone and cannot regenerate bone

without a diversity of cells in the milieu and the various

signals from growth factors and the microenvironment.

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Marx/Harrell

270 Volume 2, Number 4, 2012

Therefore, concentrating the cellular components

of whole bone marrow, rather than isolating and ex-

panding selected MSCs and/or HSCs, optimizes and

promotes both osteogenesis and angiogenesis. The

authors suggest that the use of the full diversity of hu-

man nucleated bone marrow cells and increasing their

numbers translates into enhanced bone regeneration

that is superior to outcomes obtained by single cell

expansions or single growth factor applications.

The long-awaited goal of rebuilding lost skeletal

parts of signiﬁcant size without the morbidity of open

bone harvesting (eg, a long hospital stay, a long con-

valescent period, scarring, disability, and higher costs)

has now become a reality. Although researchers have

just begun to understand the complexities of human

bone marrow cell functions and their interactions, the

value of rhBMPs, and the matrices upon which bone

can regenerate, it is known that composite grafts of

an appropriate cellular milieu, a signal, and a suitable

matrix can regenerate up to 8 cm of missing bone. The

challenge now is to expand the knowledge and un-

derstanding of adult bone marrow cells, the signal of

rhBMP, and the best matrix for bone growth to apply

these results to larger skeletal defects and those as-

sociated with a hostile tissue environment (eg, radiat-

ed tissue, scar tissue, bisphosphonate-compromised

bone).

ACKNOWLEDGMENTS

Dr Marx is a paid consultant for Harvest Technologies and

Medtronic, Inc. Dr Harrell is the chief research scientist at Harvest

Technologies.

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Table 3 Activities and Roles of the Growth Factors and the Microenvironment in a Graft

Factor Action

Hypoxia Driving force for wound healing via macrophage chemotaxis

Fibronectin and ﬁbrin (from plasma) Cell adhesion molecules; matrix for osteosynthesis

Vitronectin (from platelets) Cell adhesion molecule; matrix for osteosynthesis

Stromal-derived activating factor 1-alpha (from platelets) Homing signal for MSCs and HSCs

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MSC Differentiate into osteoblasts; bone formation; respond to HSCs and growth factors

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