Tissue-engineered bone for treatment of combat related limb injuries
Aim: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized...
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irk-123456789-1385402018-06-20T03:05:14Z Tissue-engineered bone for treatment of combat related limb injuries Vasyliev, R.G. Oksymets, V.M. Rodnichenko, A.E. Zlatska, A.V. Gubar, O.S. Gordiienko, I.M. Zubov, D.O. Original contributions Aim: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. Materials and Methods: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. Results: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn’t any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3–6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5–6 months post-op. Conclusion: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient. 2017 Article Tissue-engineered bone for treatment of combat related limb injuries / R.G. Vasyliev, V.M. Oksymets, A.E. Rodnichenko, A.V. Zlatska, O.S. Gubar, I.M. Gordiienko, D.O. Zubov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 191–196. — Бібліогр.: 26 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138540 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Vasyliev, R.G. Oksymets, V.M. Rodnichenko, A.E. Zlatska, A.V. Gubar, O.S. Gordiienko, I.M. Zubov, D.O. Tissue-engineered bone for treatment of combat related limb injuries Experimental Oncology |
| description |
Aim: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. Materials and Methods: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. Results: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn’t any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3–6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5–6 months post-op. Conclusion: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient. |
| format |
Article |
| author |
Vasyliev, R.G. Oksymets, V.M. Rodnichenko, A.E. Zlatska, A.V. Gubar, O.S. Gordiienko, I.M. Zubov, D.O. |
| author_facet |
Vasyliev, R.G. Oksymets, V.M. Rodnichenko, A.E. Zlatska, A.V. Gubar, O.S. Gordiienko, I.M. Zubov, D.O. |
| author_sort |
Vasyliev, R.G. |
| title |
Tissue-engineered bone for treatment of combat related limb injuries |
| title_short |
Tissue-engineered bone for treatment of combat related limb injuries |
| title_full |
Tissue-engineered bone for treatment of combat related limb injuries |
| title_fullStr |
Tissue-engineered bone for treatment of combat related limb injuries |
| title_full_unstemmed |
Tissue-engineered bone for treatment of combat related limb injuries |
| title_sort |
tissue-engineered bone for treatment of combat related limb injuries |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2017 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138540 |
| citation_txt |
Tissue-engineered bone for treatment of combat related limb injuries / R.G. Vasyliev, V.M. Oksymets, A.E. Rodnichenko, A.V. Zlatska, O.S. Gubar, I.M. Gordiienko, D.O. Zubov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 191–196. — Бібліогр.: 26 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T06:00:40Z |
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2025-07-10T06:00:40Z |
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| fulltext |
Experimental Oncology 39, 191–196, 2017 (September) 191
TISSUE-ENGINEERED BONE FOR TREATMENT OF COMBAT
RELATED LIMB INJURIES
R.G. Vasyliev1, 2, V.M. Oksymets2, A.E. Rodnichenko1, 2, A.V. Zlatska1, 2, O.S. Gubar2, 3,
I.M. Gordiienko2, 4, D.O. Zubov1, 2,*
1State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of Ukraine,
Kyiv 04114, Ukraine
2Biotechnology Laboratory ilaya.regeneration, Medical Company ilaya®, Kyiv 03115, Ukraine
3Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv 03680, Ukraine
4R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
Aim: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal
cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for
restoration of critical sized bone defects caused by combat related high energy trauma. Materials and Methods: To fabricate bone equivalent
we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent
mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures
was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell
phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect
treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed
by the radiographic examination. Results: Casualties were included in a treatment program an average of 10.1 months after injury, provided
the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentia-
tion potential and functional properties, ~30% colony forming units frequency and hadn’t any signs of cell senescence. The fluorescein
diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding
with viable cells. Pathomorphological analysis of bone equivalent specimens 3–6 months post-op revealed the active remodeling processes
and immature bone tissue formation. Bone defect restoration was observed 5–6 months post-op. Conclusion: The developed biotechnology
of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone
integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient.
Key Words: regenerative medicine, bone defects, tissue-engineered bone equivalent, human cell-based medicinal products, multi-
potent mesenchymal stromal/stem cells, endothelial progenitor cells.
The problems of high energy traumatic injuries
of skeleton bones and the methods of their recon-
struction in correlation with reparative osteogenesis
are relevant for actual clinical and experimental trau-
matology and orthopedics. Temporary and economic
costs for treating patients with alterations of reparative
regeneration processes, especially in the high-energy
mechanism of trauma, the complexity of their social
adaptation, justify the need to search for innovative
organ-saving technologies of regenerative medicine
for bone integrity restoration. Particularly, it is actual
the problem of treatment of combat related high energy
bullet, shrapnel and blast injuries Ukrainian traumatolo-
gists faced with, seeing the military actions in eastern
part of Ukraine.
The gold standard for extended bone defects’
treatment is autologous osteoplastic surgery. Then
there are methods of the defect filling with use of dif-
ferent osteoplastic materials, such as allogeneic and
xenogeneic bone (e.g., demineralized bone matrix);
synthetic and polymer materials; and using the method
of compression-distraction osteosynthesis by Ilizarov.
The choice of the method of plastic of the bone defect
depends on its location and size.
The newest alternative to the conventional methods
of bone plastic surgery is bone restoration methods based
on the approaches of regenerative medicine. Currently,
there are two approaches to bone regene ration. The
first one is based on the delivery of high doses of bone
morphogenetic and other factors to the defect zone,
such as autologous platelet concentrate or recombinant
bone morphogenetic proteins (BMPs). Marx et al. [1] first
evaluated the use of platelet concentrate for bone healing
in 1998, and his studies led to the development of an entire
industry for its use in traumatology and orthopedics using
platelet concentrator devices. BMPs were first proposed
as bone morphogenes by Urist in 1965 [2]. In the late
1980s his team performed the first transplantation with
their use [2, 3].
The second approach in bone regeneration is based
on the delivery to the defect area of the critical mass
Submitted: August 04, 2017.
*Correspondence: E-mail: zoubov77@yahoo.com
Tel.: +380506970600
Abbreviations used: 3D TEBE — three-dimensional tissue-engi-
neered bone equivalent; ADSCs — adipose-derived stem cells;
ALP — alkaline phosphatase; BM-MSCs — bone marrow-derived
multipotent mesenchymal stromal/stem cells; BMPs — bone mor-
phogenetic proteins; CFU — colony forming units; EPCs — endo-
thelial progenitor cells; FBS — fetal bovine serum; FDA — fluores-
cein diacetate; MSCs — multipotent mesenchymal stromal/stem
cells; PBS — phosphate buffered saline; PI — propidium iodide;
PPCs — periosteal progenitor cells.
Exp Oncol 2017
39, 3, 191–196
192 Experimental Oncology 39, 191–196, 2017 (September)
of osteoprogenitor cells, which realize their trophic and
integrative potencies in a wound for the formation of bone
regenerate and new bone at final. For the first time Phe-
mister in the 1940s used injections of bone marrow aspi-
rate into the fracture non-unions [4]. In the 1960s Burwell
proposed as an autograft of the iliac crest bone containing
the bone marrow [5]. Subsequently, as a source of os-
teoprogenitor cells, a concentrated bone marrow-derived
mononuclear fraction was used. Connolly first showed
in 1998 that four-fold concentration of the mononuclear
fraction of bone marrow significantly increases the
degree of fusion of non-consolidated fractures in hu-
mans [6]. Muschler et al. in 2003 proposed the technique
of saturation of allogeneic bone chips and crumbs with
a bone marrow clot, both in animal models and in clinical
practice of fracture consolidation [7, 8]. After preclinical
studies of Muschler’s technique, on a par with autograft
application, the positive results of its effectiveness were
confirmed by several completed multicenter clinical trials
[9, 10]. However, in some cases, neither the autograft
nor Muschler’s technique led to the formation of a dense
bone in non-consolidated fractures revealed by the X-ray
examination. Such dysfunction may be related to the
individual osteogenic insufficiency of bone marrow cells,
as well as to the compromised immune status of the pa-
tient. Thereby, the next step in bone regeneration was the
use of immunomodulatory allogeneic cultured osteopro-
genitor bone marrow-derived multipotent mesenchymal
stromal/stem cells (BM-MSCs) [11–14]. Of note, the
organ-saving bone regeneration technology Trinity Evolu-
tion™ (Orthofix, USA) based on Mushler’s approach has
been proposed to restore defects in casualties with com-
bat related trauma obtained in Iraq and Afghanistan war
conflicts. As an osteoinductive scaffold the authors from
Walter Reed National Military Medical Center (Bethesda,
USA) have used the allogeneic demineralized bone matrix
chips pre-saturated with allogeneic cadaver mononuclear
fraction of bone marrow containing uncultured and non-
purified MSCs [15]. Furthermore, in other studies for bone
fracture consolidation, it was applied an alternative cell
component — the adipose-derived MSCs, or adipose-
derived stem cells (ADSCs) [16, 17].
Considering the safety profile of cell therapies, it was
explored a recent review analyzing 70 clinical studies
worldwide with more than 1400 patients involved and
treated with autologous or allogeneic adipose-derived
MSCs. Thus, in case of allogeneic use it was shown the
generation of specific antibodies towards donor cells
in 19–34% of patients with not yet well known systemic
impact. Concerning the oncological safety, only one case
of breast cancer recurrence was noted out of 121 previ-
ous breast cancer patients within the 12-month follow-
up periods. In other three trials examined with 32 previous
prostate cancer patients involved, no cancer recurrences
were found within 3 to 6 months follow-up period [18].
Our biotechnology approach to bone integrity res-
toration is based on the transplantation of a living three-
dimensional tissue-engineered bone equivalent (3D TEBE)
developed by our R&D group since 2004 in Ukraine [19,
20]. In its last modification the advanced 3D TEBE
consists of decellularized allogeneic bone block of the
required size and shape (for circular defects), or bone
chips or crumbs (for tangential defects). These scaffolds
are used as osteoinductive and osteoconductive carriers
for cultured autologous osteoprogenitor BM-MSCs and
periosteal progenitor cells (PPCs) intended for formation
of the critical cell mass for new bone formation in situ, and
endothelial progenitor cells (EPCs) from patient’s own
peripheral blood intended to enhance the vascularization
and trophics of the manufactured 3D TEBE.
MATERIALS AND METHODS
Casualties. The forty seven casualties were included
for a moment into the experimental treatment program
assured by the Medical company ilaya® Bioethics Com-
mission permission, patient’s informed consent, local
protocol for treatment approved by the Ministry of Health
of Ukraine; 39 injured patients with 42 bone defects have
been completely treated. Selected patients hadn’t previ-
ous oncologic diseases reported and possessed circular
and tangential critical sized defects (ones without spon-
taneous recovery throughout life, more than 3.0 cm)
of tubular bones and heel bone of various lengths and
volume. The defects were allocated according to the
Table 1. Assessment of the effectiveness of a treatment
was performed by the X-ray examination over 3 and
6 months after grafting. In some cases, during the 3D
TEBE adaptive resection surgery the extra fragments
of the remodeling graft was taken, which was subse-
quently subjected to the pathomorphological analysis
(e.g., 6 months after grafting, human cells). The sections
were stained with hematoxylin & eosin.
Cell cultures
Culture of the BM-MSCs. Heparinized bone marrow
aspirate in a volume not more 5 ml was obtained with
an aspirating needle from the patient’s iliac crest. Then,
1 ml of aspirate was spread over culture flasks with an area
of 175 cm2 (SPL, Korea). The primary culture was seeded
into multi-flasks with an area of 875 cm2 (Corning, USA) for
large scale cell expansion. The therapeutic dose of BM-
MSCs (50–300•106 cells) was available over 30–40 days.
Culture of osteoprogenitor PPCs. A culture of PPCs
was obtained from a minimal fragment of the fibula
periosteum up to 1 cm3. The fragment was washed with
antibiotic/antimycotic solution (BioWest, France) and
poured into the 100 mm Petri dish (SPL, Korea). The
primary culture was seeded into multi-flasks with an area
of 875 cm2 (Corning, USA). The therapeutic dose of PPCs
(20–60•106 cells) was available over 30 days.
The growth medium for BM-MSCs and PPCs cultur-
ing consists of α-MEM, 10% fetal bovine serum (FBS),
1 ng/ml bFGF (Sigma-Aldrich, USA), 2 U/ml heparin
sodium (Indar, Ukraine), 100 U/ml antibiotic-antimycotic
solution (BioWest, France). Cell subculturing was car-
ried out with a mixture of trypsin and EDTA solutions
in a ratio of 0.05%:0.02% in phosphate buffered saline
(PBS) (Sigma-Aldrich, USA). The seeding density was
103 cells per 1 cm2.
Culture of EPCs from the peripheral blood. EPCs were
obtained from 20 ml of heparinized venous blood of the
Experimental Oncology 39, 191–196, 2017 (September) 193
patient. Each 5 ml blood was spread per 75 cm2 culture
flask (SPL, Korea) into the selective endothelium growth
medium EGM-2 (Lonza, Italy). Single EPCs colonies from
the primary culture were seeded in culture flasks with
an area of 175 cm2 (SPL, Korea) to obtain a therapeutic
dose. Cell subculturing was carried out with a mixture
of trypsin and EDTA solutions in a ratio of 0.1%:0.02%
in PBS (Sigma-Aldrich, USA). The seeding density was
3•103 cells per 1 cm2. The therapeutic dose of EPCs
(20–60•106 cells) was available over 30 days.
All of the above cell types were grown in a multi-gas
incubator (Binder CB 210, Germany) in an atmosphere
of 5% CO2 and 5% O2 and a saturating humidity of 97%.
All patients (peripheral blood — by immunofluores-
cence assay, polymerase chain reaction) and finalized
cell cultures (by polymerase chain reaction) were
screened for absence of HIV ½, HBV, HCV, HSV ½,
CMV, EBV, Treponema pallidum and Mycoplasma ssp.
Functional assays for quality control. A set of qual-
ity control procedures were established to release per-
sonalized 3D TEBE as the human cell-based medicinal
product. To determine the cell plating efficiency, colony
forming units (CFU) analysis was performed according
to the conventional protocol. For CFU staining, the cell
colonies were fixed with cold ethanol and stained with
azure-eosin by Romanowsky (Makrokhem, Ukraine) for
20 min [21]. Multilineage differentiation into the osteo-
genic and adipogenic directions was carried out according
to the standard protocols [22]. Alkaline phosphatase (ALP)
detection was carried out by the BCIP/NBT substrate as-
say (Sigma-Aldrich, USA). The normal karyotype of cul-
tured cells was confirmed by GTG banding method [21].
Directed multilineage differentiation assays
Adipogenic differentiation was performed in the
following medium: DMEM-HG (4.5 g/l) supplemented
with 10% FBS, 1 µM dexamethasone, 200 µM indo-
methacine, 500 µM isobutylmethylxanthine, 5 µg/ml in-
sulin (Sigma-Aldrich, USA) and 5% donor horse serum
(BioWest, France). After 14 days the cells were fixed and
stained. To detect the adipogenic differentiation, the
cells were stained with 0.5% solution of Oil Red O dye
for neutral intracellular lipids (Sigma-Aldrich, USA) [22].
Osteogenic differentiation was performed in the follow-
ing medium: DMEM low glucose (1.0 g/l) with 10% FBS,
100 nM dexamethasone, 10 mM β-glycerophosphate and
50 µg/ml ascorbate-2-phosphate (Sigma-Aldrich, USA).
After 21 days the cells were fixed and stained. To detect
osteogenic differentiation, the cells were stained with 2%
solution of Alizarin Red S dye for calcified extracellular
matrix deposition (Sigma-Aldrich, USA) [22].
Endothelial cell tube formation assay. The assay
measures the ability of endothelial cells, plated at sub-
confluent densities with the appropriate extracellular
matrix support, to form capillary-like structures (tubes).
The EPCs were added to the Matrigel™ Matrix-coated
(Corning, USA) well of 24-well plate (SPL, Korea) at the
density 103 cells per 200 µl, at a final medium volume
of 200 µl per 1 cm2 per well. Then they were incubated
at 37 °C, 5% CO2, 5% O2 overnight in selective endothe-
lium growth medium EGM-2 (Lonza, Italy). EPCs develop
well-formed tube networks within 4–6 hours [23].
Flow cytometry. The number of positive and
negative cells for corresponding markers (conven-
tional, but not all-sufficient set) [14, 22] was mea-
sured for cultured BM-MSCs, PPCs and EPCs (at P2,
n = 5 per cell type) with use of flow cytometer BD FACSAria
(BD Biosciences, USA). Staining with the monoclonal
antibodies (PerCP-Cy5.5 mouse anti-human CD105,
APC mouse anti-human CD73, FITC mouse anti-human
CD90, PerCP-Cy5.5 mouse anti-human HLA-DR,
PE-Cy-7 mouse anti-human CD31, PE mouse anti-human
CD271, APC mouse anti-human CD34, FITC mouse anti-
human CD45) was performed according to the manufac-
turer’s instructions (BD Pharmingen, BD Horizon, USA).
3D TEBE manufacturing. As a scaffold for cells,
a treated, cell-, DNA-free, allogeneic and non-immu-
nogenic bone blocks of the required size and shape,
bone chips and bone crust were used (A.A. Partners
Ltd., Ukraine). In a first step the carrier was seeded
directly with cells; in a second step the cell-seeded
carrier was placed into the fibrin-derived hydrogel, also
containing the cells. Cells were seeded over a carrier
and into the fibrin hydrogel at a ratio of 2–5•106 cells per
1 cm3 scaffold. If the bone defect did not exceed 3 cm,
then the equivalent with only cultured BM-MSCs was
used; if more than 5 cm — BM-MSCs and PPCs were
used at a ratio of 3:1; if more than 7 cm — BM-MSCs,
PPCs and EPCs were applied in a ratio of 3:1:1. The ap-
propriately seeded 3D TEBE before the transplantation
was incubated for 5–14 days according to the size and
volume in the BM-MSCs growth medium in a multi-gas
incubator (Binder CB 210, Germany) in an atmosphere
of 5% CO2 and 5% O2 and a saturating humidity of 97%.
The quality and regularity of seeding of the equivalent
fragment with cells were confirmed by the combined
staining with fluorescein diacetate/propidium iodide
(FDA/PI) fluorescent dyes (Sigma-Aldrich, USA).
Microscopy. Inverted fluorescent microscope
AxioObserver A1 equipped with digital camera AxioCa-
mERc 5s and ZEN 2012 software (Carl Zeiss, Germany)
were applied.
Statistics. The data are presented as mean and
SEM (M ± m). Statistical significance was estimated
using the Student’s t-criterion.
RESULTS AND DISCUSSION
The gold standard for critical sized bone defect
restoration is autologous bone graft transplantation.
Although this surgery possesses its drawbacks, such
as morbidity, risk of donor wound contamination and
non-healing, risk of lysis of transplanted bone autograft.
As a substantial alternative for autologous bone grafting
we developed the regenerative medicine organ-saving
biotechnology of the 3D TEBE transplantation.
The total number of treated casualties using tissue
engineering approach was 47 (male — 46, female —
1). The treatment was completed in 39 patients. Forty
seven wounded persons had critical sized bone de-
fects of different location (Table 1). Eight patients are
194 Experimental Oncology 39, 191–196, 2017 (September)
in the treatment course. The age of the patients was
21–48 years (the average age is 33.9 years). Before
entering the clinic course, the wounded persons were
subjected to 2–27 surgeries (an average of 5.7 sur-
geries). The patients came in treatment 2–22 months
(an average of 10.1 months) after the combat related
injury and unsuccessful surgeries in other foreign and
Ukrainian hospitals. Most casualties had received a pre-
scription to the limb amputation.
Table 1. Total number of patients and allocation of critical sized bone de-
fects in casualties subjected to 3D TEBE transplantation
Bone defect
allocation Arm Forearm Femur Shin Heel Total
n %
Proximal third 4 − 2 6 − 12 25.5
Diaphysis 5 5 8 10 − 28 59.6
Distal third 3 − − 1 − 4 8.5
Calcaneus − − − − 3 3 6.4
Total 12 5 10 17 3 47 100.0
Transplantation of 3D TEBE in the form of bone
blocks was performed in 10 wounded, in the form
of chips — in 22 wounded, and in the form of bone
blocks and chips — 13 casualties. The volume of the
transplant ranged from 10 to 180 cm3 (on the average
40.4 cm3). The average density of graft seeding was
4.7 million cells per 1 cm3. The manufactured bone
equivalent was homogenously seeded with cells after
incubation on a scaffold as proved by FDA/PI combined
staining procedure. The cell cultures were appropriately
characterized for their identity, purity, potency, viability
and suitability for the intended use [14, 22, 23]. All cell
cultures had a normal karyotype and phenotype, dif-
ferentiation potential and functional properties, ~30%
CFU frequency and hadn’t any signs of cell senescence.
Cultured BM-MSCs and PPCs were positive for
conventional (but not unique) stromal markers CD105,
CD90, CD73, negative (< 2%) by hematopoietic markers
CD34, CD45, and negative for MHC class II molecules —
HLA-DR (Table 2). Cultured PPCs were also positive for
CD271 (a versatile pre-culture marker, reported to be ex-
pressed only on isolated, but not cultured adult MSCs
to selectively isolate and expand the adult stromal cells
with both immunosuppressive and lympho-hematopoietic
engraftment-promoting properties) [24]. Cultured EPCs
were highly positive for CD31 (key endothelial marker),
CD105 and CD73, positive for CD34 (~35%), and negative
for CD45 and HLA-DR. It is interesting to observe the ex-
pression of the CD90 stromal marker on in vitro expanded
EPCs being low as 3.4% (see Table 2).
Table 2. Positive and negative cell surface markers of all cultured cell
types involved in 3D TEBE manufacturing (determined by flow cytometry).
Data presented as a percentage of parent population, %
Cell cul-
ture CD105 CD90 CD73 CD271 CD31 CD34 CD45 HLA-
DR
BM-MSC 99.7 ±
0.1
95.6 ±
1.2
99.8 ±
0.0
ND ND 0.4 ±
0.1
0.4 ±
0.1
0.8 ±
0.3
PPCs 97.5 ±
1.7
97.7 ±
1.4
99.4 ±
0.4
29.7 ±
3.7
ND 1.1 ±
0.6
0.3 ±
0.1
1.6 ±
0.6
EPCs 99.2 ±
0.3
3.4 ±
1.4
99.6 ±
0.2
ND 99.2 ±
0.1
35.0 ±
11.7
0.7 ±
0.2
2.7 ±
1.2
Note: at P2, n = 5; ND — not determined.
Of note, only PPCs, but not BM-MSCs cultures
at their basal culture level were ALP positive. Moreover,
the PPCs cultures failed their adipogenic differen-
tiation assay after 14 days of induction. It means the
strong osteogenic commitment of this cell type without
directed osteogenic induction in vitro (Fig. 1).
Histological analysis of 3D TEBE biopsies taken
during graft adaptation resection surgery 3–6 months
after transplantation showed graft intensive remodel-
ing and immature bone tissue formation (Fig. 2).
Restoration of the bone defects was observed after
5–6 months post-op and evaluated by radiographic ex-
amination in 39 patients (42 critical sized bone defects).
The results of the treatment are done in the Table 3 and
considered as the following: Good — the formation
Fig. 1. Cell cultures’ functional assays for QC: a — BM-MSCs
(morphology, immunophenotype, karyotype, directed diffe-
rentiation assay); b — PPCs (morphology, immunophenotype,
directed differentiation assay, BCIP/NBT for ALP detection); c —
EPCs (morphology, immunophenotype, karyotype, endothelial
cell tube formation assay in Matrigel™ Matrix)
Experimental Oncology 39, 191–196, 2017 (September) 195
of bone tissue with the restoration of the integrity of bone
segments of a limb within 4–6 months post-op (Fig. 3);
Satisfactory — patients who had partial graft lysis in the
transplantation area, but bone was formed provided the
functional capacity of injured limb; patients who expe-
rienced a delay (more than 6 months) for bone tissue
formation post-op; Unsatisfactory — patients who had
a complete lysis of the transplanted bone equivalent.
Thereof, the overall effectiveness of treatment of critical
sized bone defects with 3D TEBE was 90.4%.
Table 3. The results of completed critical sized bone defects’ treatment
with use of 3D TEBE
Results n %
Good 30 71.4
Satisfactory 8 19.0
Unsatisfactory 4 9.6
Total 42 100
Note: 39 patients with 42 critical sized bone defects.
The biomedicinal product based on the human
cells developed by our group can be attributed,
depending on the main regulatory systems (Euro-
pean Medicines Agency — Committee for Advanced
Therapies, EU vs Food and Drug Administration, USA)
either to the (1) “Human Cell-Based Medicinal Prod-
ucts” — in terms of the European Medicines Agency
[25], or to the (2) “Human cells, tissues, and cellular
and tissue-based products”, those in terms of the
Food and Drug Administration [26]. Seeing the signing
of the Association agreement between Ukraine and the
European Union in 2014, our legislation and regulatory
on the transition of biomedicinal product based on hu-
man cells into the clinical practice should be gradually
harmonized with the EU legislation.
Based on our developed method for treatment
of critical sized bone defects with the use of re-
generative medicine approaches, we have planned
and registered the clinical study at the International
Registry ClinicalTrials.gov entitled “3D Tissue Engi-
neered Bone Equivalent for Treatment of Traumatic
Bone Defects (3D TEBE)”; ClinicalTrials.gov Identifier:
NCT03103295. ClinicalTrials.gov is a registry and re-
sults information database of clinical research studies
sponsored or funded by a broad range of public and
private organizations around the world [27].
Thereof, the developed regenerative medicine
approach to organ-saving transplantation of the liv-
ing 3D TEBE, with the overall effectiveness of 90.4%,
allows restoring the bone integrity, forming new bone
tissue in a site of bone defect of critical size, and sig-
nificantly reducing the rehabilitation period of a patient.
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