Tissue Engineering Bone - Reconstruction of Critical Sized Segmental Bone Defects in a Large Animal Model Johannes Christian Reichert, MD Faculty of Built Environment and Engineering, School of Engineering Systems, Queensland University of Technology Thesis submitted for: Doctor of Philosophy (PhD) 2010
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Tissue Engineering Bone - Reconstruction of
Critical Sized Segmental Bone Defects in a
Large Animal Model
Johannes Christian Reichert, MD
Faculty of Built Environment and Engineering, School of Engineering
Chapter IV – Establishment of a Preclinical Ovine Model for Tibial
Segmental Bone Defect Repair by Bone Tissue
Engineering Methods
115
- Introduction 117
- Regulatory framework 119
- Intramedullary nail versus plate fixation versus external
fixator
124
- Road map to establish a preclinical model for segmental bone
defect research
128
- Pilot study limited contact locking compression plate
(LC-LCP)
129
- Finite element modelling
131
- Implant testing 132
- Pilot study dynamic compression plate 134
- Summary 137
Chapter V – Reconstructing large segmental bone defects in an ovine
model by tissue engineering methods
139
- Introduction 141
- Materials and Methods 145
- Scaffold fabrication and preparation 145
- Anaesthesia and pre-operative treatment 147
- Defect model 146
- Harvest of autologous cancellous bone graft 150
- Experimental groups 151
- Euthanasia 151
- Radiographic analysis 152
- Computed tomography 152
- Biomechanical testing 154
IX
- !CT analysis 155
- Histology 157
- Statistical analysis 157
- Results 158
- Animal model 158
- X-ray analysis 158
- Computed tomography 160
- Biomechanical testing 165
- !CT analysis 168
- Histology 173
- Discussion 176
- Summary 185
Conclusions and Recommendations 187
Bibliography 191
X
XI
List of illustrations and diagrams
Chapter I
Figure 1: PMMA sections of ovine tibial bone demonstrating secondary
osteon formation Table 1: Factors influencing the size of a critical sized defect Table 2: Bone biomechanical properties of different species Table 3: Overview of segmental bone defect studies Table 4: Comparison of animal models for fracture and segmental bone
defect research Table 5: Summary of human and large animal bone properties
Chapter II
Figure 1: Mesenchymal progenitor cell and osteoblast isolation and
expansion Figure 2: Uncoated and collagen I coated polycaprolactone tricalcium-
over seven days in monolayer Figure 4: Surface antigen expression of ovine mesenchymal progenitor
cells and osteoblasts Figure 5: Clonogenic efficiency of ovine mesenchymal progenitor cells
and osteoblasts Figure 6: Alizarin red, osteocalcin, and type I collagen staining of
mesenchymal progenitor cell and osteoblast monolayers after 28 days of osteogenic induction
Figure 7: Quantification of alizarin red incorporated in mesenchymal
progenitor cell and osteoblast monolayer cultures after 28 days of osteogenic culture
Figure 8: Quantification of alkaline phosphatase activity at day 14 and 28
of osteogenic culture in monolayer
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Figure 9: Quantitative RT-PCR for osteogenic markers after 28 days of culture under osteogenic conditions in monolayer
Figure 10: Alkaline phosphatase activity under static and dynamic
monolayer culture conditions Figure 11: Alizarin red quantification at day 14 of static and dynamic
osteogenic monolayer cultures Figure 12: Calcium amounts in dynamic cultures after 14 days Figure 13: XPS analysis of ovine bone and Thermanox coverslips Figure 14: XPS analysis of extracellular matrix synthesized by ovine
mesenchymal progenitor cells and osteoblasts Figure 15: Proliferation of mesenchymal progenitor cells and osteoblasts
under static and dynamic conditions Figure 16: Scanning electron microscopy, FDA-PI and Phalloidin-DAPI
staining of mesenchymal progenitor cells and osteoblasts cultured on mPCL-TCP scaffolds for 28 days
Figure 17: MicroCT analysis of mesenchymal progenitor cell and
osteoblast 3D cultures on collagen I coated polycaprolactone tricalcium-phosphate scaffolds
Figure 18: Histology, histomorphometry and microCT analysis of
transplanted in vivo specimens after eight weeks
Chapter III
Figure 1: Schematic illustrating the in vitro generation of a transplantable
tissue engineered construct Figure 2: Immunohistochemical staining for BrdU of an ovine
tissue engineered constructs after eight weeks Figure 4: 3D microCT reconstructions of subcutaneously transplanted
tissue engineered constructs after eight weeks Figure 5: Bone volume fractions and bone mineral density of
transplanted tissue engineered constructs after eight weeks as determined by microCT analysis
XIII
Figure 6: H&E staining on paraffin sections of cell free mPCL-TCP scaffolds that were transplanted alone, in combination with fibrin glue or with fibrin glue containing rhBMP-7
Figure 7: H&E staining on paraffin sections of mesenchymal progenitor
cell or osteoblast seeded mPCL-TCP scaffolds that were transplanted in combination with or without rhBMP-7
Figure 8: Von Kossa/van Gieson staining on PMMA sections of cell free
mPCL-TCP scaffolds that were transplanted alone, in combination with fibrin glue or with fibrin glue containing rhBMP-7
Figure 9: Histomorphometric analysis of mineralized tissue within
transplanted tissue engineered constructs Figure 10: Histomorphometric analysis of neovascularisation within
transplanted tissue engineered constructs Figure 11: Von Kossa/van Gieson staining on PMMA sections of
mesenchymal progenitor cell or osteoblast seeded mPCL-TCP
scaffolds that were transplanted in combination with or without
rhBMP-7
Figure 12: High magnification of PMMA sections stained for von
Kossa/van Gieson showing differences in morphology of bone lining cells
Figure 13: Immunohistochemistry for osteocalcin on paraffin sections Figure 14: Alcian blue staining on paraffin sections demonstrating
glycosaminoglycan deposits Figure 15: Immunohistochemistry for type II collagen on paraffin sections Figure 16: Histochemical staining for tartrate resistant acid phosphatase
on paraffin sections illustrating osteoclast activity Figure 17: Immunohistochemical staining and histomorphometry for BrdU
labelled cells on paraffin sections Figure 18: Backscattered scanning electron microscopy and Energy
dispersive X-ray spectroscopy
XIV
Chapter IV
Figure 1: Flow chart describing a road map to establish a critically sized defect model
Figure 2: Schematic of commonly applied methods for segmental defect
fixation Figure 3: Image showing a segmental tibial defect of 2 cm length
stabilized with a LC-LCP Figure 4: Image of different implants chosen for biomechanical testing
and set up of the four point bending test Figure 5: Equivalent bending stiffnesses of tested implants Figure 6: Image illustrating a segmental tibial defect of 2 cm length
stabilized with a DCP, postoperative radiograph and x-ray after 12 week
Table 1: Advantages and disadvantages of different fixation devices
Chapter V
Figure 1: 3D microCT reconstruction of a cylindrical mPCL-TCP scaffold
of 3 height and 2cm diameter Figure 2: Image series demonstrating the application of and OP-implant
to a cylindrical mPCL-TCP scaffold Figure 3: Intraoperative images of the critical size defect creation in an
ovine tibia Figure 4: Image series illustrating the harvesting procedure of autografts
from the iliac crest Figure 5: 3D CT reconstruction of a 3 cm tibial defect overlayed with a
developed CT scoring system Figure 6: DICOM image of an intact ovine tibia (axial view)
Figure 7: Embedding procedure for torsional testing Figure 8: Schematic illustrating the method of calculation for the polar
moment of inertia Figure 9: Illustration of the cutting planes for histological sectioning
XV
Figure 10: Radiographs demonstrating bone formation within defects of
the different experimental groups after 12 weeks
Figure 11: Bar graphs demonstrating union rates and determined CT
scores for the different experimental groups
Figure 12: 3D CT reconstructions of representative specimens of each
experimental group
Figure 13: Box plots demonstrating the total bone volumes and newly
formed bone in the cortical region determined by quantitative
analysis of CT scans
Figure 14: Bone volumes determined for the marrow and external callus
regions determined by quantitative analysis of CT scans
Figure 15: Box plot illustrating torsional moment values measured for the
different experimental groups
Figure 16: Box plot showing torsional stiffness values calculated for the
different experimental groups
Figure 17: MicroCT sections and 3D microCT reconstructions of
representative samples of the different experimental groups
Figure 18: Box plots demonstrating the volume of newly formed bone
within the defects determined by quantitative microCT analysis
Figure 19: Bone volume distribution along the z axis of the 3 cm defects
Figure 20: Box plots showing the tissue mineral density of the newly
formed bone within the 3 cm defects
Figure 21: Box plots illustrating the trabecular thicknesses for proximal,
medial and distal defect portions and thickness distribution
along the z axis.
Figure 22: Box plots demonstrating the polar moment of inertia calculated
for the 3 cm defect regions of the different experimental groups
Figure 23: Histological sections of the entire 3 cm defects of each group
stained for Safranin O/von Kossa and Movat’s pentachrome
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Figure 24: Higher magnification images of histological defect sections
stained for Movat’s pentachrome showing the different
composition of tissue formed within the 3 cm defects
Figure 25: High magnification images of PMMA sections stained for
Movat’s pentachrome illustrating the different degrees of bone
maturation within the different experimental groups
XVII
List of abbreviations
2D Two dimensional 3D Three dimensional ABG Autologous bone graft ALP Alkaline phosphatase AO Arbeitsgemeinschaft osteosynthese ARC Australian Research Council bFGF Basic fibroblast growth factor BMP Bone morphogenetic protein BrdU 5-bromo-2-deoxyuridine BSA Bovine serum albumin BV Bone volume CAD Computer aided design CD Cluster of differentation CFU Colony forming unit CO2 Carbon dioxide CSD Critical sized defect DAPI 4',6-diamidino-2-phenylindole DCP Dynamic compression plate DMEM Dulbecco’s modified eagle media DNA Deoxyribonucleic acid ECM Extracellular matrix EDTA Ethylene-di-amine-tetra-acetic acid EDX Energy dispersive X-ray
spectroscopy EU European union f frequency FACS Fluorescence activated cell sorting FBS Foetal bovine serum FDA Fluorescein diacetate FDA Food and drug administration FDM Fused deposition modelling FEM Finite element modelling FGF Fibroblast growth factor FITC Fluorescein isothiocyanate GAGs Glycosaminoglycans GDF Growth differentiation factor H2O water HCl Hydrogen chloride HE Haematoxylin-eosin HGF Hepatocyte growth factor IGF-1 Insulin like growth factor 1 ISO International organization for
and peri-operative management strategies have procured better treatment
outcomes of complex fractures and other skeletal defects caused by high
energy trauma, disease, and tumours [1-6]. However, a compromised wound
environment, biomechanical instability and other factors can result in large
defects with limited intrinsic regeneration potential [7]. Such defects pose a
major surgical, socio-economical and research challenge, and highly
influence patients’ quality of life [8, 9].
Cancellous bone fractures of the proximal humerus, distal radius, or the tibial
plateau can lead to bone impaction and consequently defect formation after
reduction [4]. The tibial diaphysis, however, represents the most common
anatomic site for segmental bone defects since it is devoid of muscle
coverage on its anteromedial surface [8]. This poor soft tissue coverage both
increases the risk of bone loss and complicates treatment [8].
Over the years, bone autografts have advanced as the “gold standard”
treatment to augment and accelerate bone regeneration [1, 2, 10-16]. The
application of autografts, however, is associated with considerable negative
side effects. Graft harvest leads to prolonged anaesthetic periods and
requires personnel [12, 14, 17]. Often, insufficient amounts of graft can be
obtained while the access to donor sites is limited [12, 13, 18, 19]. Donor site
morbidity (persistent pain and haemorrhage) is common, the risk of infection
is increased, and the transplanted bone is predispositioned to failure [4, 12,
8
13, 20]. Graft failures usually result from incomplete transplant integration,
particularly in large defects [14]. In addition, graft devitalisation due to
insufficient graft vascularisation and subsequent resorption processes can
lead to decreased mechanical stability [21].
The transplantation of vascularised autografts is time consuming and
technically demanding. Allografts and xenografts carry the risk of immune-
mediated rejection, graft sequestration, and transmission of infectious
disease [9, 22-28]. The dense nature of cortical bone allografts impedes
revascularization and cellular invasion from host sites following implantation
[18]. This limited ability to revascularize and remodel is believed to account
for an allograft associated failure rate of 25% and a complication rate of 30-
60% [18, 29]. In addition, the maintenance of bone banks is associated with
considerable operating expenses.
A technique introduced to avoid graft integration-related difficulties known as
the “Ilizarov technique”, involves the osteotomy of bone combined with
distraction to stimulate bone formation. This procedure has been applied
successfully to treat large bone defects, infected non-unions, and limb length
discrepancy [30]. However, the Ilizarov technique is a long-lasting procedure,
inconvenient for the patient [31, 32], and recurrent pin track infections are a
frequent complication [24, 33].
In order to avoid the limitations associated with the current standard
treatment modalities for segmental bone deficiencies, research efforts have
focused on the use of naturally derived and synthetic bone graft substitutes
during the past decades.
9
More recently, the concept of tissue engineering has emerged as an
important approach to bone regeneration related research. Tissue
engineering unites aspects of cellular biology, biomechanical engineering,
biomaterial sciences, and trauma and orthopaedic surgery. Its general
principle involves the association of cells with a natural or synthetic
supporting scaffold to produce a three-dimensional, implantable construct.
Introduction
To biomechanically simulate human in vivo conditions as closely as possible,
and to assess the effects of implanted bone grafts and tissue engineered
constructs on segmental long bone defect regeneration, a number of large
animal models have been developed. However, most of the preclinical
models published are not well described, defined or standardized. In 2008,
the Journal of Bone and Joint Surgery published a number of review papers
on preclinical models in fracture healing and non-unions [34]. However,
these articles provide only rudimentary information on how to establish
relevant segmental bone defects in a preclinical large animal model. Hence,
the following chapter provides detailed, comprehensive information on the
advantages and disadvantages of the different published animal models.
Definition of a Critical-Sized Bone Defect
It has been postulated that an experimental osseous injury inflicted to study
bone repair mechanisms needs to be of dimensions to preclude spontaneous
healing [35]. Therefore, the non-regenerative threshold of bone was
determined in different research animal models inducing so-called “critical
10
sized” defects. These critical sized defects are defined as “the smallest size
intraosseous wound in a particular bone and species of animal that will not
heal spontaneously during the lifetime of the animal” [29, 36, 37] or as a
defect which shows less than ten percent bony regeneration during the
lifetime of an animal [37].
Table 1: Factors influencing the size of a critical defect
The minimum size that renders a defect ‘‘critical’’ is not well understood. For
practical reasons, it has been defined as a segmental bone deficiency of a
length exceeding 2-2.5 times the diameter of the affected bone [24, 33].
Results of various animal studies suggest that critical sized defects in sheep
however, could even be approximately three times the diameter of the
corresponding diaphysis [33]. Nevertheless, a critical defect in long bones
cannot simply be defined by its size, but also depends on the species
phylogenetic scale, anatomic defect location, associated soft tissue, and
biomechanical conditions in the affected limb as well as age, metabolic and
systemic conditions, and related co-morbidities affecting defect healing
(Table 1)[24, 36].
Factors determining a CSD [24] [29, 36]
o Age o Species phylogeny o Defect size o Anatomic location o Bone structure and vascularisation o Presence of periosteum o Adjacent soft tissue o Mechanical loads and stresses on the limb o Metabolic and systemic conditions o Fixation method/stiffness o Nutrition
11
Large animal models in bone defect research
Animal models in bone repair research include representations of normal
fracture-healing, segmental bone defects, and fracture non-unions, in which
regular healing processes are compromised in the absence of a critical-sized
defect site [38]. In critical-sized segmental defect models, bridging of the
respective defect does not occur despite a sufficient biological
microenvironment as critical amounts of bone substance are removed. In
contrast, in true non-unions, deficient signalling mechanisms, biomechanical
stimuli or cellular responses may prevent defect healing rather than the
defect size.
When selecting a specific animal species as a model system, a number of
factors need to be considered. With comparison to humans, the chosen
animal model should clearly demonstrate both significant physiological and
pathophysiological analogies in respect to the scientific question under
investigation. Moreover, it must be manageable to operate and observe a
multiplicity of study objects over a relatively short period of time [39-41].
Further selection criteria include costs for acquisition and care, animal
availability, acceptability to society, tolerance to captivity, and ease of
housing [42].
Over the last decades, several publications have described dogs as a
suitable model for research related to human orthopaedic conditions [43]. It
was found that dogs closely resemble humans with regards to bone weight,
density and bone material constituents such as hydroxyproline, extractable
proteins, IGF-1, organic, inorganic and water fraction although clear
differences in bone microstructure and remodelling have been described [44,
12
45]. While the secondary structure of human bone is predominantly
organized into osteons, osteonal bone structure in dogs is limited to the core
of cortical bone, whereas in areas adjoining the periosteum and endosteum
mainly laminar bone is found as characteristic for large, fast-growing animals
[46]. It has been reported that generally, higher rates of trabecular and
cortical bone turnover can be observed in dogs compared to humans [47]
and differences in loads acting on the bone, as a result of the dog’s
quadrupedal gait must also be taken into consideration as well. Various
biomechanical properties as described in table 2.
Bone biomechanical properties
Cortical bone
Trabecular bone
Dog Humerus (bending) E: 2.66 (GPa) UStress 193.23 (MPa) [48]
and porosity to allow cell and blood vessel infiltration into the carrier and
inflammatory tissue reactions might be few reasons. New delivery systems
with optimized and controlled release profiles may hence decrease or even
alleviate the need for excessive and expensive concentrations of BMPs.
As in the present study, segmental bone defects usually heal via indirect
repair mechanisms referred to as endochondral ossification. This process
involves the recruitment, proliferation, and differentiation of mesenchymal
progenitor cells into cartilage, which subsequently becomes calcified and
eventually is replaced by bone. The repair process is comprised of four
overlapping phases initiated by an immediate inflammatory response that
leads to the recruitment of mesenchymal progenitor cells and subsequent
differentiation into chondrocytes that produce cartilage and osteoblasts,
183
which form bone. After cartilage matrix is produced, it mineralizes, and a
transition from mineralized cartilage to bone occurs, initiated by the
resorption of mineralized cartilage [269]. It is the eventual bridging of hard
callus areas across the central defect gap that provides the initial
stabilization and regain of biomechanical function [270]. Primary bone
formation is followed by remodelling, in which the initial bony callus is
reshaped by secondary bone formation and resorption to restore the
anatomical structure that supports mechanical loads [271].
The current study results suggested not only a tendency towards increased
bone formation in defects treated with rhBMP-7 but also stimulated callus
maturation as evidenced by fewer amounts of osteoid and mineralized
cartilage in this group compared to autograft treated defects. These findings
could be attributed to rhBMP-7 accelerating the molecular events associated
with defect healing. Bone defect repair recapitulates the molecular pathways
of normal embryonic development with the coordinated participation of
several cell types originating from the cortex, periosteum, surrounding soft
tissue, and bone marrow space. The signalling molecules regulating this
process include pro-inflammatory cytokines, the transforming growth factor-
beta (TGF-") superfamily and other growth factors, and the angiogenic
factors [271, 272]. The transforming growth factor-beta (TGF-ß) superfamily
consists of a large number of growth and differentiation factors that include
bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-
ß), growth differentiation factors (GDFs), activins, inhibins, and Mullerian
inhibiting substance. Specific members of this family - such as BMPs (2-8),
GDF (1, 5, 8 and 10), and TGF-ß 1-3 - promote various stages of
184
intramembranous and endochondral bone ossification during bone healing
[273].
Recent studies demonstrate that BMP-7 specifically induces the expression
of numerous growth factors and multiple members of the BMP family
(including autoinduction), during the bone induction process [274, 275].
These factors then guide the bone formation process to completion.
Expression of BMP-7 was found to be enhanced in sites of endochondral
ossification about one week after fracture and it was therefore suggested that
BMP-7 acts predominately in the early stage of fracture healing [276]. In vitro
studies have shown that rhBMP-7 perpetuates mechanisms involved in bone
formation, including the promotion of chondrocyte maturation (extracellular
matrix component production) in normal articular chondrocytes, the
enhancement of osteoblastic characteristics (alkaline phosphate production)
of normal osteoblast cells, and the induction of the differentiation of
osteoclasts involved in the bone remodelling process. The end result of this
differentiation cascade is the production of weight-bearing bone with fully
functional bone marrow elements [277].
Considering the role of BMP-7 in mainly early stages of defect healing
together with the described release kinetics of BMP from collagen carriers,
and the favourable properties of the applied scaffolds, one could conclude
that in the present study, the requirements - based on the current knowledge
surrounding bone defect healing - have been met as closely as possible to
produce a suitable tissue engineered bone graft substitute.
185
Summary
In the current study, a standardized and reproducible ovine, tibial, critical-
sized defect model was developed as reflected by the non-union rate of
100% in the empty control group. The application of autografts from the iliac
crest caused defect bridging in all cases. When compared to intact controls,
the mechanical properties of the newly formed bone however were of inferior
quality after 3 months. Medical grade PCL-TCP scaffolds did not evoke
foreign body responses. The mPCL-TCP scaffolds alone did not result in any
substantial mineralization of the defect area. However, when combined with
rhBMP-7, bone formation within the defect equalled or even exceeded
amounts observed with autografts. The study results suggest that mPCL-
TCP scaffolds combined with a biologically active stimulus such as rhBMP-7
can serve as an equivalent alternative to autologous bone grafting in the
early phase of defect regeneration. These findings however must be
confirmed by long-term studies.
186
187
Conclusions and Recommendations
The detailed characterisation of cells involved in bone regeneration
associated processes is of utmost importance. Since sheep represent a
valuable model for human bone turnover and remodelling, data
characterising cells derived from these animals are of special interest. Ovine
marrow derived mesenchymal progenitor cells and cortical bone osteoblasts
exhibit morphological, immunophenotypical and multipotential characteristics
similar to those in humans, which underlines the value of sheep as a model
species. Unfortunately, available methods of analysis are limited as ovine
genes are only partially sequenced and very few antibodies specific for and
cross-reacting with equivalent sheep antigens are available. If further insight
into fundamental processes such as haematopoiesis, cell migration and
homing, injury repair, differentiation, and proliferation is to be provided, these
shortcomings need to be addressed in the future.
In vitro experiments identified mesenchymal progenitor cells reproducibly
displaying a higher osteogenic potential. In vivo, however, osteoblasts
exhibited a higher potential to form new bone. These results emphasize the
difficulties in extrapolating in vitro findings to in vivo settings and suggest that
osteoblasts isolated from compact bone possibly represent a suitable
alternative cell population for cell based tissue engineering applications.
When comparing the osteogenic potential of these cells after transplantation
in vivo, subcuntaneously transplanted cells showed a high degree of survival
and actively contribute to endochondral osteogenesis. Endochondral bone
188
formation, which describes a process in which a cartilage template is
gradually replaced by a bone matrix. It can also be observed in orthotopic
segmental defect sites. When compared to mesenchymal progenitor cells,
osteoblasts deposited higher amounts of new bone while osteoblast derived
bone was of higher maturation. Cell stimulation with rhBMP-7 increased the
rate of bone synthesis for both cell types and positively affected
neovascularisation and osteoclast activity. These results suggest that origin
and commitment of transplanted cells can determine type and degree of
ossification. They furthermore confirm that rhBMP-7 represents a potent
adjuvant stimulating bone formation.
It needs to be emphasized that microenvironmental conditions in ectopic
transplantation sites, again, may not be representative of specific cues cells
experience in a large segmental bone defect. However, it could also be
argued, that in such a defect cells are - similarly to ectopic sites - surrounded
by mainly soft tissue types rather than bone. Although an essential first step
was taken towards the characterization of ovine mesenchymal progenitor
cells and osteoblasts, essentially, further studies are required to verify these
findings in orthotopic models.
In the present study, the most important issues related to the establishment
of a large preclinical model for segmental bone defect research have been
addressed and discussed, and it was demonstrated how to develop such a
model. Regarding bench to bedside translations, an important milestone was
achieved in establishing a highly reproducible large animal model as an
189
essential prerequisite to systematically assess different bone grafting
materials, scaffolds, tissue engineered constructs and growth factors.
The performance of a novel tissue engineered construct was finally assessed
in a large animal model and compared to the standard autograft
transplantation. The application of autografts from the iliac crest caused
defect bridging in all cases. When compared to intact contralateral controls,
the mechanical properties of the newly formed bone, however, were of
inferior quality. Transplanted scaffolds showed good biocompatibility and did
not evoke foreign body responses. They did, however, not result in any
substantial mineralization of the defect area. However, when combined with
BMP-7, bone formation within the defects equalled or even exceeded
amounts observed with autografts. Therefore, the study results suggest that
these scaffolds combined with a biologically active stimulus such as BMP-7
can serve as an equivalent alternative to autologous bone grafting in the
early phase of defect regeneration. These findings however must be
confirmed by long-term studies (12-24 months) that also assess the events
surrounding scaffold degradation and bone remodelling. Lastly, if this novel
and promising technology is to be translated into a routine clinical application
with predictable outcomes stringent treatment indications/contraindications
need to be formulated and detailed dose-effect and dose-response
relationships determined taking into account variables such as defect size
and cause or patient age.
190
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