Bone grafting is an integral part of reconstructive surgery. In the United States alone over 250,000 bone grafts were harvested annually. While autogenic bone grafting has always been associated with donor site morbidity, bone graft substitutes have been suggested as a solution. In this project, a bone graft substitute using human dental pulp cells (HDPCs) and peptide nanofibre hydrogel was being developed.
HDPCs were isolated from extracted teeth. After culture and expansion, unsorted HDPCs were encapsulated into peptide nanofibre hydrogel. These cell-gel constructs were cultured for two weeks in ordinary culture medium and then for 2-3 more weeks in osteogenic lineage induction medium. The post-induced cell-gel derived constructs were transplanted into skin pouches or calvarial bone defects of nude mice.
When transplanted subcutaneously, the cell-gel derived constructs were harvested at four to twelve weeks postoperatively (n=5). Tissue samples were processed for contact radiograph, histological examination and antibody staining. These constructs developed into vascularised, mineralised tissue pieces. Though bone marker proteins (osteopontin, osteocalcin and osteonectin) were detected in these tissue pieces, the histological structure of their tissue matrix did not resemble bone matrix.
Later, it was accidentally noted that portions of constructs touching the bone defect margin, would form bone through direct matrix transformation. This indicated that the current cell-gel model was potentially the first study model of tissue engineering bone by simulating intramembranous ossification.
In the bone defect trial, obviously mineralized cell-gel derived constructs of matching shape and size were selected to patch the 3mm calvarial bone defects (n=5). Bone defect specimens were harvested at two weeks post-operation. The development of radio-opaque structures within the bone defects were evaluated in virtual 3-dimensional models constructed with data collected by microtomographic scanning. The bone nature of these radio-opaque structure were validated histologically (by staining with Hematoxylin and Eosin, Periodic acid-Schiff stain and Picrosirius red) and immunologically (with antibody against human collagen-I, osteonectin and parathyroid hormone receptor). The radio-opaque structures developed into the bone defect were evaluated positive for bone. And significantly more bone regeneration was observed in the test group (n=4) than in the control (n=2). The mean area percentages of regeneration were 46.3% and 0% respectively (p< 0.05).
While the majority of studies in bone tissue engineering have worked with bone marrow stromal cells and scaffolds of synthetic polymer or calcium based materials, this is the first successful attempt of using HDPCs and peptide nanofibre hydrogel to engineer bone (in a nude mice mode). And the potential of these cell-gel derived constructs to promote bone regeneration was demonstrated.
But this was the result from a single experiment of small sample size in one animal model only. It needs to be fortified by further experiments with larger population size and in other animal models.
To increase clinical usefulness, the construct will need to be scaled up to centimetre size level. This will necessitate a change of its configuration from bead into meshwork. And, the data collected to date will shed light onto the redevelopment of all the relevant protocols. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/188268 |
| Date | January 2013 |
| Creators | Chan, Kwok-ming., 陳國明. |
| Contributors | Wong, RWK |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B50533873 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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