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Cell delivery systems for bone tissue engineering

Evi Lippens UGent (2009)
abstract
Bone is a very active and dynamic tissue that is constantly remodelled in response to mechanical stress and hormonal stimuli. The cells of the bone are capable of restoring bone defects and fractures in their original state. However, in certain circumstances, the spontaneous repair process is hampered, resulting in non-unions and/or the formation of fibrous scar tissue. When the repair process of the bone fails, additional treatment is needed to aid the healing and to preserve the integrity of the bone. The standard treatment consists of transplanting bone grafts in the defects. These grafts can be harvested from the patient (autograft) or from other human (allograft) or animal (xenograft) origin. Autologous bone grafting is still the golden standard treatment, but as with allograft and/or xenograft treatment, several drawbacks have been reported. The search for the ideal treatment for bone defects is ongoing to optimise the bone repair process. Especially the field of bone tissue engineering receives a lot of attention. Tissue engineered implants are constructs of a biomaterial (scaffold, carrier) and bioactive factors. These factors can be cells and/or growth factors that stimulate the hosts repair response. The goal is to mimic as closely as possible the organization, functionality and structure of bone. It was the aim of this thesis to find a new tissue engineered construct (TEC) to augment the bone repair process. This has been carried out in close collaboration with the Polymer Chemistry and Biomaterials Research Group of Prof. Etienne Schacht for the fabrication of new biomaterials and with the group of Prof. Frank Gasthuys from the Department of Surgery and Anaesthesiology of Domestic Animals for the evaluation of the TEC in an animal model. Since most bone defects are irregular in shape, it is difficult to fit in a preformed scaffold. That is why a scaffold that is formed inside the defect by an in situ polymerization technique is preferred. Moreover, the biomaterial can be delivered in a minimal invasive way through injection. Cells, i.e. bone marrow derived mesenchymal stem cells (BMSC), can be mixed with the biomaterial prior to injection. Cells were first cultured on a porous carrier system to offer the cells protection during the mixing in and the curing of the biomaterial and to offer these anchorage dependent cells a substrate for attachment and proliferation. Two types of in situ forming materials were investigated i.e. a poly(D,L-lactide-co-ε-caprolactone) polyester with a cross-linkable methacrylate end group and a UV cross-linkable chemically modified form of the Pluronic® F127 hydrogel with N-methacryloyl depsipeptide end groups. The porous carrier systems were on the one hand the commercially available gelatine microcarrier CultiSpher-S® and on the other hand a hydroxyapatite (HA) carrier developed at VITO. Implantation of cell loaded CultiSpher-S® carriers mixed in the lactide-caprolactone polyester in unicortical tibia defects in goats showed the survival of the cells on the encapsulated carriers, but there was no formation of new bone originating from the added cell source. The limited porosity of the polymer combined with the slow degradation rate of the polymer hampered the ingrowth of new blood vessels and bone. As an alternative, a chemically modified form of the FDA approved Pluronic® F127 hydrogel was tested in vitro and in vivo in the same goat model. There were clear indications of new bone formation originating from the implanted cells on the CultiSpher-S® carriers. Furthermore there was a general tendency of better bone formation in comparison to the natural bone healing capacity of the untreated control defect. However when the cells were cultured on the HA carriers and encapsulated into the same modified hydrogel, there was no formation of newly formed bone originating from the cells on the HA carriers. Further research is needed to find an explanation for these unexpected results with the HA carriers. Finally, the possibility of cryopreservation of cell loaded carriers was investigated so that the in vitro culture time before implantation could be decreased. Different mixtures of cryoprotective agents were tested for their contribution to cell survival. Each mixture contained dimethyl sulphoxide (Me2SO) either in a 10vol% concentration or in 5vol% concentration with 5vol% of a non-penetrating cryoprotectant. Best results were obtained after cryopreservation in the presence of 10vol% Me2SO. Cryopreservation of BMSC and MC3T3-E1 preosteoblasts cultured on CultiSpher-S® carriers showed immediately after thawing a decline in cell viability but the same level of cell colonization on the carriers as before cryopreservation was obtained after an additional three days of culture. This study indicates that cryopreservation of cell loaded CultiSpher-S® carriers is possible and could indeed reduce the in vitro culture time prior to implantation.
Please use this url to cite or link to this publication:
author
promoter
UGent and UGent
organization
year
type
dissertation (monograph)
subject
keyword
bone tissue engineering, construct cryopreservation
pages
VIII, 166 pages
publisher
Ghent University. Faculty of Medicine and Health Sciences
place of publication
Ghent, Belgium
defense location
Gent : UZ (auditorium C)
defense date
2009-12-09 17:30
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted materials
copyright statement
I have transferred the copyright for this publication to the publisher
id
1114548
handle
http://hdl.handle.net/1854/LU-1114548
date created
2011-02-02 09:42:24
date last changed
2011-02-03 11:21:50
@phdthesis{1114548,
  abstract     = {Bone is a very active and dynamic tissue that is constantly remodelled in response to mechanical stress and hormonal stimuli. The cells of the bone are capable of restoring bone defects and fractures in their original state. However, in certain circumstances, the spontaneous repair process is hampered, resulting in non-unions and/or the formation of fibrous scar tissue. When the repair process of the bone fails, additional treatment is needed to aid the healing and to preserve the integrity of the bone. The standard treatment consists of transplanting bone grafts in the defects. These grafts can be harvested from the patient (autograft) or from other human (allograft) or animal (xenograft) origin. Autologous bone grafting is still the golden standard treatment, but as with allograft and/or xenograft treatment, several drawbacks have been reported. The search for the ideal treatment for bone defects is ongoing to optimise the bone repair process. Especially the field of bone tissue engineering receives a lot of attention. Tissue engineered implants are constructs of a biomaterial (scaffold, carrier) and bioactive factors. These factors can be cells and/or growth factors that stimulate the hosts repair response. The goal is to mimic as closely as possible the organization, functionality and structure of bone. 
It was the aim of this thesis to find a new tissue engineered construct (TEC) to augment the bone repair process. This has been carried out in close collaboration with the Polymer Chemistry and Biomaterials Research Group of Prof. Etienne Schacht for the fabrication of new biomaterials and with the group of Prof. Frank Gasthuys from the Department of Surgery and Anaesthesiology of Domestic Animals for the evaluation of the TEC in an animal model. 
Since most bone defects are irregular in shape, it is difficult to fit in a preformed scaffold. That is why a scaffold that is formed inside the defect by an in situ polymerization technique is preferred. Moreover, the biomaterial can be delivered in a minimal invasive way through injection. Cells, i.e. bone marrow derived mesenchymal stem cells (BMSC), can be mixed with the biomaterial prior to injection. Cells were first cultured on a porous carrier system to offer the cells protection during the mixing in and the curing of the biomaterial and to offer these anchorage dependent cells a substrate for attachment and proliferation. Two types of in situ forming materials were investigated i.e. a poly(D,L-lactide-co-\ensuremath{\epsilon}-caprolactone) polyester with a cross-linkable methacrylate end group and a UV cross-linkable chemically modified form of the Pluronic{\textregistered} F127 hydrogel with N-methacryloyl depsipeptide end groups. The porous carrier systems were on the one hand the commercially available gelatine microcarrier CultiSpher-S{\textregistered} and on the other hand a hydroxyapatite (HA) carrier developed at VITO.
Implantation of cell loaded CultiSpher-S{\textregistered} carriers mixed in the lactide-caprolactone polyester in unicortical tibia defects in goats showed the survival of the cells on the encapsulated carriers, but there was no formation of new bone originating from the added cell source. The limited porosity of the polymer combined with the slow degradation rate of the polymer hampered the ingrowth of new blood vessels and bone. 
As an alternative, a chemically modified form of the FDA approved Pluronic{\textregistered} F127 hydrogel was tested in vitro and in vivo in the same goat model. There were clear indications of new bone formation originating from the implanted cells on the CultiSpher-S{\textregistered} carriers. Furthermore there was a general tendency of better bone formation in comparison to the natural bone healing capacity of the untreated control defect. However when the cells were cultured on the HA carriers and encapsulated into the same modified hydrogel, there was no formation of newly formed bone originating from the cells on the HA carriers. Further research is needed to find an explanation for these unexpected results with the HA carriers.
Finally, the possibility of cryopreservation of cell loaded carriers was investigated so that the in vitro culture time before implantation could be decreased. Different mixtures of cryoprotective agents were tested for their contribution to cell survival. Each mixture contained dimethyl sulphoxide (Me2SO) either in a 10vol\% concentration or in 5vol\% concentration with 5vol\% of a non-penetrating cryoprotectant. Best results were obtained after cryopreservation in the presence of 10vol\% Me2SO. Cryopreservation of BMSC and MC3T3-E1 preosteoblasts cultured on CultiSpher-S{\textregistered} carriers showed immediately after thawing a decline in cell viability but the same level of cell colonization on the carriers as before cryopreservation was obtained after an additional three days of culture. This study indicates that cryopreservation of cell loaded CultiSpher-S{\textregistered} carriers is possible and could indeed reduce the in vitro culture time prior to implantation.},
  author       = {Lippens, Evi},
  keyword      = {bone tissue engineering,construct cryopreservation},
  language     = {eng},
  pages        = {VIII, 166},
  publisher    = {Ghent University. Faculty of Medicine and Health Sciences},
  school       = {Ghent University},
  title        = {Cell delivery systems for bone tissue engineering},
  year         = {2009},
}

Chicago
Lippens, Evi. 2009. “Cell Delivery Systems for Bone Tissue Engineering”. Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
APA
Lippens, E. (2009). Cell delivery systems for bone tissue engineering. Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium.
Vancouver
1.
Lippens E. Cell delivery systems for bone tissue engineering. [Ghent, Belgium]: Ghent University. Faculty of Medicine and Health Sciences; 2009.
MLA
Lippens, Evi. “Cell Delivery Systems for Bone Tissue Engineering.” 2009 : n. pag. Print.