Page 350 - Read Online

and osteogenesis properties. [1,2] However, its limitations METHODS
include additional surgical exposure required for graft
harvest, limited bone supply and associated donor site Bone marrow mesenchymal stem cells isolation
morbidities. [3,4] Vascularized bone grafts from various and expansion
locations including the fibula, scapula and iliac crest may Bone marrow mesenchymal stem cells (BM‑MSCs) were isolated
be indicated to stimulate bone formation and promote from rabbits and cultured as reported previously. [9,11,13,15] The
healing. However, harvest requires a complex microsurgery BM aspirates (5 mL) were obtained from the femurs of
procedure, with the additional risk of including graft 5 rabbits that were 8 weeks old. The BM aspirates were
necrosis due to vessel thrombosis. [5,6] Allografts may be a layered over a Ficoll gradient and centrifuged at 2,000 rpm
reasonable alternative, as small cancellous allografts can for 20 min at room temperature. Mononuclear cells at
remodel completely. Larger grafts may be incorporated the interface were collected and cultured in Dulbecco’s
by limited intramembranous bone formation. However, Modified Eagle Medium‑Low glucose supplemented with
[1]
allografts may increase the risk of infectious disease 15% fetal bovine serum (FBS), 10 mM L‑glutamine, 10 ng/mL
transmission. epidermal growth factor, 10 ng/mL β‑fibroblast growth factor
Recent progress in the fields of biotechnology and and 1% antibiotic antimycotic solution (10,000 U/mL
tissue engineering has offered new options for the penicillin, 10 mg/mL streptomycin and 25 µg amphotericin B)
repair of traumatic and nontraumatic bone defects. (Sigma‑Aldrich, USA). Cultures were maintained in a CO
2
Mesenchymal stem cells (MSCs), which are multipotent incubator (Shel Lab, USA) at 37 °C and 5% CO . The medium
2
adult stem cells of mesodermal origin, have been shown was replaced after 3 days and cell layers were washed
to play a critical role in tissue engineering. MSCs are twice with phosphate‑buffered saline to remove nonadherent
an excellent potential source of cells for bone tissue cells. The passage was carried out when cultures reached 90%
engineering due to their excellent renewal ability and confluence using 0.25% trypsin‑ethylenediaminetetraacetic
osteogenic differentiation capabilities. [7,8] In addition to acid (Sigma‑Aldrich, USA). Cell confluence was normally
the bone marrow (BM), MSCs are also derived from the achieved after 12‑14 days. [14,16‑18]
periosteum. It is well known that the development and Scaffold materials
regeneration of bone depend on the presence of periosteum Human demineralized cancellous bone (HDCB), which
and BM. When transferred to the site of bone damage, is a type of DBM and has been proven to be usable as
[9]
MSCs multiply and differentiate into osteoblastic cells, scaffold material, was used in this experiment. HDCB was
contributing to the production of bone tissues that form supplied from the Bone Bank at the National Institute of
a callus at the bone defect site. Alternatively, bone Burns. Fresh bones were aseptically harvested within the
[10]
tissue engineering can be achieved via intramembranous first 12 h after being shown to be free of any infectious
ossification. [11] disease. Bones were treated with H O , a mixture of
2
2
The use of MSCs with an appropriate scaffold has methanol/chloroform, hydrochloric acid and phosphate
been demonstrated to be promising in guiding bone buffer pH 7.4. Subsequently, the bones were dehydrated
tissue neoformation after implantation in the host. Cell for 24 h until the water content remaining in the bones
repopulation can be achieved either by direct cell loading was less than 5%. The bones were cut into blocks with
or indirect cell induction with osteogenic factors. [12,13] dimensions of 1.5 cm × 0.3 cm × 0.5 cm. A medullary
Combining MSCs with appropriate scaffolds has been hole was made in the bone blocks with a diameter of
shown to improve the overall osteoconductivity of the 1.5 mm. The block was packaged and sterilized by gamma
scaffold. The search for an ideal scaffold has led to the irradiation at a dose of 25 kGy. The sterilized bones were
development of reconstructive options to engineer then preserved at 4 °C.
new bone (NB) tissue. The ideal scaffold should be Tissue engineered bone graft preparation in vitro
biocompatible, noninfectious, resorbable, osteoconductive Culture‑expanded BM‑MSCs were seeded evenly onto the
and osteoinductive. Demineralized bone matrix (DBM), HDCB scaffold. DHCB/BM‑MSCs were cultured in T flasks
[14]
which is derived from either allogenic or xenogenic (Thermo Scientific Nunc A/S, Denmark) filled with 5 mL
bone, is available commercially for clinical application DMEM containing 10% FBS and antibiotics. The grafts
and satisfies some of these requirements. DBM has were placed in a vacuum desiccator and treated at a
[4]
been used for several decades in humans for the pressure of 100 torr for 100 s, after which they were
treatment of nonunion and bone defects following injury incubated at 37 °C, 5% CO for 2 weeks. The medium was
or tumor resection. The process of demineralization replaced every 3 days. [2,19‑21] 2
using hydrochloric acid destroys potential bone forming
agents, but also decreases antigenic stimulation and may Animals and surgical procedure
expose the bone morphogenic protein located within the Twenty‑eight male 8‑week‑old white New Zealand rabbits
bone matrix. [1,4] This study is designed to evaluate the with a body weight of approximately 1.5 kg from the
bone formation properties of a demineralized cancellous Experimental Laboratory of the Medical Training and
bone scaffold seeded with allogenic MSCs, with or Research Center, Hue Central Hospital, were used for
without periosteum, in a critical sized bone defect model the study. All the experimental study involving animals
in rabbits. was approved by the Institutional Animal Care and Use

Plast Aesthet Res || Vol 2 || Issue 6 || Nov 12, 2015 341