International Orthopaedics (SICOT)
DOI 10.1007/s00264-016-3130-6
ORIGINAL PAPER
A method of treatment for nonunion after fractures
using mesenchymal stromal cells loaded on collagen
microspheres and incorporated into platelet-rich plasma clots
Olga Wittig 1 & Egidio Romano 1 & Cesar González 2 & Dylana Diaz-Solano 1 &
Maria Elena Marquez 1 & Pedro Tovar 2 & Rodolfo Aoun 2 & Jose E. Cardier 1
Received: 1 January 2016 / Accepted: 8 February 2016
# SICOT aisbl 2016
Abstract
Purpose There is evidence showing that mesenchymal stro-
mal cells (MSC) may constitute a potential therapeutic strate-
gy to induce bone regeneration. In this work, we investigate
the capacity of autologous bone marrow (BM) MSC loaded
on collagen microspheres (CM) and included into autologous
platelet-rich plasma (PRP) clots (MSC/CM/PRP) to induce
bone formation in patients with nonunion lesions.
Methods MSC were isolated from BM cells of patients with
nonunion lesions. Phenotypical (marker expression) and func-
tional studies (osteogenic differentiation) were performed.
MSC were seeded on CM and included into autologous PRP
clot (MSC/CM/PRP). The capacity of MSC/CM/PRP to in-
duce bone formation was evaluated in three patients diag-
nosed with nonunion.
Results MSC loaded on CM/PRP clots maintain their biolog-
ical functions, in vitro. After three months, post-MSC trans-
plantation, all patients showed evidence of osteogenesis at the
site of nonunion. After one year, all patients showed a com-
plete healing of the nonunion.
* Egidio Romano
egidio_romano@yahoo.es
* Jose E. Cardier
jcardier@ivic.gob.ve
1
Unidad de Terapia Celular - Laboratorio de Patología Celular y
Molecular, Instituto Venezolano de Investigaciones Científicas
(IVIC), Apartado 21827, Caracas 1020-A, Venezuela
2
Servicio de Traumatología, Hospital Universitario de Caracas,
Caracas 1080, Venezuela
Conclusions Our results support the use of autologous MSC
transplanted as MSC/CM/PRP for the treatment of nonunion
fractures. Future studies incorporating a larger number of pa-
tients may confirm the results obtained in this work.
Keywords Bone regeneration . Collagen . Mesenchymal
stromal cells . Nonunion . PRP
Introduction
Nonunion constitutes one of the most serious complica-
tions of bone fracture. Although the aetiology of non-
union is not clearly understood, there is evidence show-
ing that excessive mechanical instability of the fracture
and a reduction of bone vascularity are involved in the
pathogenesis of this disease [1–3]. Different kinds of
treatments have been used to repair the bone defects
associated with nonunion [4, 5]. However, most of them
are associated with morbidities, limited supply, frequent
treatment failure, and high cost [4–6].
Mesenchymal stromal cells (MSC) constitute a popu-
lation of cells with a multipotential capacity of differen-
tiation into bone, cartilage, fat, and endothelial cells
(among others) [7–10]. It is known that MSC play a
key role in the process of bone formation and fracture
repair [3, 8, 9, 11, 12]. Based on this knowledge, the
use of MSC as a potential therapeutic strategy to induce
formation of new bone tissue and fracture healing is an
area of intense research in regenerative medicine. In this
study we evaluated the bone regeneration capacity of
autologous BM-MSC, seeded on collagen microspheres
(CM) and included into autologous platelet-rich plasma
(PRP) (MSC/CM/PRP), in patients with nonunion.
 International Orthopaedics (SICOT)

Fig. 1 Culture, characterization,

and differentiation of MSC.
Adherent cells from BM show
fibroblast-like cell morphology
(a) and express MSC markers (b).
MSC differentiate into
osteogenic, chondrogenic, and
adipogenic progenitors (c, d, e,
respectively); and form vessel like
structures (f, arrows)

Material and methods
Reagents Monoclonal antibodies anti-human CD34,
CD45, CD14, CD90, CD73, CD29, CD49b, CD166
were from BD Biosciences (USA). Microspheres
atellocollagen and collagen sponge were purchased from
Cosmo Bio (Tokyo, Japan).
Patients Three patients with nonunion fractures, which had not
responded to orthopedic surgery, were enrolled in this study.
Informed consent was obtained from all individual participants
and the study protocol was approved by the Bioethics Committee
of Hospital Universitario de Caracas. This study was performed
in accordance with the ethical standards as laid down in the 1964
Declaration of Helsinki and its later amendments.

Fig. 2 Functionality of MSC on

CM. CM maintained in culture
(a). Adhesion, proliferation (head
arrows), migration (arrows) (b),
and osteogenic differentiation of
MSC on CM (c)
International Orthopaedics (SICOT)
Table 1 Clinic characteristics of
patients with nonunion
Age
Diagnosis
Time of evolution
Number of transplanted MSC
Isolation and culture of human BM-MSC MSC were iso-
lated from BM aspirates obtained from the posterior
iliac crest of each patient. MSC were cultured with
alpha-MEM-Chang medium (Irvine Scientific, USA)
supplemented with 20 % autologous serum (regular me-
dium). Bacterial, mycotic, and karyotype tests were per-
formed before MSC transplant. Cellular processing was
performed at the cellular therapy unit of our institutions,
under GMP conditions.
Phenotypic and functional analysis Expression of MSC
(CD90, CD73, CD166, CD29, and CD49b) and hematopoiet-
ic (CD34, CD45, and CD14) markers was performed by flow
cytometry. The multipotential capacity of MSC was examined
by culturing these cells in secondary cultures using osteogen-
ic, chondrogenic, adipogenic, and endothelial differentiation
media [9, 10].
Transplant of MSC in patients After three to four weeks
in culture, MSC were harvested and incubated separate-
ly in osteogenic medium and in endothelial medium for
three days. One day before operation, the cells were
seeded on CM, and in the operating room, the MSC/
CM preparation was mixed with autologous platelet-rich
plasma (PRP). Clots were obtained by adding 5 %
CaCl2/thrombin to the MSC/CM/PRP complex. Finally,
MSC/CM/PRP clots were implanted at the site of non-
union and a collagen sponge was used to cover and
close off the fracture site.
Functional studies of MSC into CM/PRP clots A sample
of a MSC/CM/PRP clot, implanted in each patient, was
evaluated for MSC growth, migration, and osteogenic
Fig. 3 MSC transplantation in patients with nonunion. MSC/CM/PRP clots (a) implanted around the nonunion site (b and c, white arrows), and a
collagen membrane placed over the nonunion site (d)
Patient 001 Patient 002 Patient 003
81 27 43
Nonunion of tibia/fibula Nonunion of of femur Nonunion of tibia
2 years 1 year 2 years
50 × 106 60 × 106 60 × 106
differentiation, following the methodology described
above.
Results
Isolation, culture and characterization of MSC Adherent
cells from BM cultures showed a typical fibroblastic
morphology (Fig. 1a) and constitutively expressed the
typical MSC markers CD90, CD166, CD73, CD29,
and CD49b (Fig. 1b). Osteogenic, chondrogenic, and
adipogenic progenitors were evidenced by alizarin red
(Fig. 1c), alcian blue (Fig. 1d), and oil red staining
(Fig. 1e), respectively. Endothelial cells were evidenced
by the formation of tubular structures resembling vessels
(Fig. 1f).
Culture of MSC on CM and included in PRP clots (MSC/
CM/PRP) We used CM (Fig. 2a) as scaffolds for MSC. Dense
areas of proliferating MSC with the formation of CM clusters
were observed (Fig. 2b). Additionally, MSC migrated out
from the CM (Fig. 2b) and differentiated into osteogenic pro-
genitors (Fig. 2c).
Transplant of MSC/CM/PRP in patients with nonunion
Three patients with nonunion were included in this
study (see Table 1). All patients had more than nine
months with the nonunion lesion. They were previously
treated with various orthopedics surgical treatments (i.e.,
osteotomy, internal, and external fixation), which failed
to cure the nonunion. Fibrotic tissue was removed from
the nonunion sites, MSC/CM/PRP clots were implanted
in the site of nonunion (Fig. 3a, b, and c), and a
 International Orthopaedics (SICOT)

collagen membrane was placed over the nonunion site infection or ectopic bone formation was observed in
containing MSC/CM/PRP (Fig. 3d). No inflammation, patients included in this study.
 International Orthopaedics (SICOT)
ƒFig. 4 Radiographic evaluation of patients after MSC transplantation.
Patient 001 showed a bone defect at the lower end of the tibia and two
defects in the fibula (pre-implant; white arrows). After three months,
evidence of osteogenesis was only observed at the tibia and fibula sites
were MSC were transplanted (arrows; no MSC/CM/PRP clots were
implanted in the upper defect of the fibula, head arrow). Three years
after MSC transplantation, the nonunion fracture completely healed.
Patient 002 showed a nonunion bone defect in the femur (pre-implant,
white arrow). MSC/CM/PRP clots were implanted in the bone defect of
the femur and it was fixed with external fixation. After six months,
evidence of osteogenesis was observed at the nonunion site (arrows).
One year after MSC transplantation, the nonunion fracture completely
healed. Patient 003 showed a nonunion bone defect at the lower end of
the tibia (pre-implant, white arrow). MSC/CM/PRP clots were implanted
at the nonunion defect and the tibia was fixed with external fixation. After
four months of MSC transplantation, osteogenesis was observed at the
nonunion site (arrows). Two years after MSC transplantation, the
nonunion fracture completely healed
Transplant of MSC/CM/PRP induces bone formation
in patients with nonunion
Case 1
An 81 years old patient (patient 001) with a single fracture of
the tibia and two fractures in the adjacent region of the fibula
(Fig. 4). Multiple surgeries were performed in this patient,
including an osteotomy with placing an external tutor to keep
the fracture in apposition (Fig. 4, pre-implant). MSC/CM/PRP
were implanted in the nonunion sites of the tibia and fibula.
MSC were not implanted at the proximal nonunion fracture of
the fibula, because most of the cells were used in the other
nonunion lesions. After three months of MSC/CM/PRP im-
plant, a broad radiopaque area was observed in the nonunion
site where the MSC were implanted with the exception of the
upper fibula fracture (Fig. 4). After three years, the tibia non-
union completely healed and the patient recovered its whole
functional capacity to normally walk.
Case 2
A 27 years old patient (patient 002) who developed a nonunion
lesion at the distal third of the femur, as a consequence of
trauma (Fig. 4). After two years of evolution and multiple or-
Fig. 5 Proliferation, migration,
and osteogenic differentiation of
MSC implanted in patients.
Aliquots of MSC/CM/PRP
implanted in each patient were
cultured (Fig. 4a). MSC in the
CM/PRP complex proliferated (b,
arrows) and differentiated into
osteogenic progenitors (c)
thopaedic surgery, including osteotomy with internal fixation
(Fig. 4, pre-implant), the patient showed the persistency of non-
union in the affected limb. MSC/CM/PRP was implanted, after
osteotomy and fibrotic tissue removal, at the site of nonunion.
An external fixation system was placed to keep the fracture in
apposition. After six months, radiological studies showed the
presence of osteogenic areas at the site where MSCs were im-
planted (Fig. 4). Fourteen months after the MSC cellular trans-
plantation, the nonunion completely healed and the patient re-
covered its whole functional capacity to normally walk.
Case 3
A 43 years old patient who developed a nonunion at the lower
third of the tibia, as a consequence of trauma. After two years
of evolution and multiple orthopaedic surgery, without con-
solidation of the fracture, including an osteotomy with placing
an external tutor (Fig. 4, pre-implant), MSC/CM/PRP was
implanted at the site of nonunion. An external fixation system
was placed to keep the fracture in apposition. After 4 months,
osteogenic areas, at the site where MSCs were implanted,
were observed (Fig. 4). After two years, the tibiae nonunion
healed and the patient recovered its whole functional capacity
and walks normally.
In vitro migration and differentiation of MSC from CM/
PRP complex A sample of MSC/CM/PRP complex
transplanted to the patients was cultured in osteogenic medi-
um. MSC adhered, migrated, proliferated, and differentiated
into osteogenic progenitor cells, in the CM/PRP complex
(Fig. 5a, b, and c).
Discussion
Although several cell therapeutic strategies for inducing bone
regeneration have been used in animal models and patients
[12, 13], there are still currently no consistent results demon-
strating the regenerative capacity of MSC in patients with
nonunion. In this work, we investigated the capacity of

MSC, seeded on CM and included in PRP clots, to induce
bone formation in three patients diagnosed with nonunion.
All patients treated with MSC/CM/PRP in this study
showed new bone formation after three to five months post-
transplantation, as assessed by radiography studies of the af-
fected segments. We did not observe any abnormal growth in
situ or adverse effect attributable to the MSC implantation,
supporting previous data about the safety of the local admin-
istration of autologous MSC [11, 13]. We believe that an im-
portant aspect of our novel treatment for bone regeneration is
the combination of MSC cultured in osteogenic and endothe-
lial differentiation media, which may provide both the capac-
ity of new bone and blood vessel formation [14]. Additionally,
the PRP clot provided not only a vehicle for the progenitor
cells but also growth signals derived from platelet [15].
In summary, our study, even if it could be considered pre-
liminary because of the small number of patients treated, shows
that transplantation of autologous MSC loaded on CM and
included in PRP may constitute a potential effective treatment
for bone regeneration in cases of nonunion secondary to frac-
tures. MSC loaded on CM and incorporated into PRP clots
maintain their biological functions, in vitro. Studies in progress
incorporating a larger number of patients may confirm the re-
sults obtained in this work, allowing the use of MSC as first line
therapy for bone defects associated with nonunion.
Acknowledgments This work was supported by funds from
FONACIT (Fondo Nacional de Ciencia, Tecnología e Investigación),
Project No. G-2005000405, and LOCTI Project No. 2011000906
Compliance with ethical standards
Conflict of interest Authors declare that they do not have any conflict
of interest to disclose.
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