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Project

Bone tissue engineering: Properties of periosteal cells vary according to their origin

Bone grafts are commonly used in orthopedics, traumatology, and oral and maxillofacial surgery for treating bone defects caused by trauma, oncologic resection, congenital malformations, and prosthetic treatments. There are still limitations and complications associated with the current standard of using autologous bone tissue as grafts. These include donor site morbidity, inadequate quantities of available donor tissue, ultimate poor tissue integration, and risk of complications as a consequence of donor tissue withdrawal. Therefore, the field is intensively investigating alternative bone graft materials.

Bones have multiple functions, providing structure, stability, and mobility with retention of flexibility via joints, to the body. The skeleton also protects internal organs, has endocrine functions and bones contain bone marrow. Bone can consist of compact cortical bone and less dense trabecular bone tissue, both of which contain mineralized tissue, osteocytes and bone-lining cells. Osteoblasts are responsible for depositing collagen and mineralizing the bone, while osteoclasts are involved in bone resorption. Bone tissue, depending on its location in the embryo and the adult body, forms through one of these two processes: intramembranous ossification (IO) and endochondral ossification (EO). IO involves the differentiation of skeletal stem and progenitor cells (SSPCs) into osteoblasts, leading to the deposition of bone matrix and subsequent vascularization of the bone. EO involves the formation of cartilage as an intermediate tissue, with SSPCs differentiating into chondroblasts that produce a cartilage matrix. The chondrocytes then become hypertrophic and subsequently either undergo cell death and allow invasion of osteoprogenitor cells and blood vessels, or they transdifferentiate into osteoblasts. Long bones are primarily formed through EO, and bone fracture healing recapitulates this process to a large extent.

Bone tissue engineering aims to provide an alternative treatment option for bone defects. It is using engineered constructs composed of appropriate cells, polypeptide growth/differentiation factors, and a 3-D environment. This approach is not yet giving totally satisfactory results required to enter the clinic. It needs to select the best source of appropriate progenitor cells, which will be combined with scaffolds, and the cells ideally can be expanded ex vivo whilst retaining their bone-forming properties. Both natural polymers and synthetic polymers have been considered for scaffold materials, and polypeptide growth/differentiation factors, particularly bone morphogenetic proteins (BMPs), are already being used to promote cell differentiation towards bone-forming cells. BMPs are multi-potent embryonic and adult cytokines that act in soft tissues of the embryo and the adult, but also play a role in bone formation. BMP2 and BMP6 direct human periosteum-derived cells towards bone formation. Engineering bone tissue via IO has limitations, since the lack of sufficiently speedy vascularization is a major hurdle, which could result in tissue necrosis. EO, via its cartilage intermediate is more adapted to a hypoxic environment and has shown promising results for bone tissue engineering. Craniofacial bones, such as those in the face and jaws, have different embryonic origins and bone formation mechanisms compared to long bones. Craniofacial bones differ in terms of their turnover rate, expression of osteoblast marker genes, and their potency of tissue mineralization. Cells that are multi-potent, particularly SSPCs, are commonly used for bone tissue engineering due to both their proliferation and differentiation capabilities. SSPCs can be obtained from various sources, including bone marrow, adipose tissue, dental pulp, and periosteum.

In this PhD thesis, the focus is primarily on periosteal-derived cells (hPDCs) including the bone-lining cells found in the maxillary sinus, and on their potential for bone tissue repair when combined with bone-promoting factors and bone-supporting biomaterials. The present study combines expertise in hPDCs, biomaterials, systems biology and transcriptomics to characterize and explore hPDCs for future clinical use. In the first part, hPDCs are isolated from different craniofacial areas (maxilla, mandible) and from the tibia, respectively. We hypothesized that these cells may have intrinsic differences and display varying bone-forming potential. We show they each have comparable proliferative capacities in cell culture, but their transcriptome, including of genes that are involved in skeletal system development, differs. Ectopic in vivo testing for de novo bone formation shows that mandibular-derived hPDCs have – both in vitro and in vivo – differentiation potentials towards cartilage and bone tissue, and present a strong potential for bone formation through EO.

In a separate, smaller project, similar investigations were conducted using cells from the Schneiderian membrane, which are bone-lining cells found in the maxillary sinus. While the Schneiderian membrane cells show good proliferative capacities and in vitro differentiation capabilities towards chondrogenic and osteogenic lineages, they were found in vivo to be less successful in bone formation compared to mandibular and tibial hPDCs.

Another separate objective was to replace the used fetal bovine serum (FBS) containing cell culture medium by a xenogeneic-free medium. For this, we tested chemically defined media (CDM) and medium supplemented with human platelet lysate (hPL). While the CDM tested for this thesis showed no successes because of excess cell death, the replacement of FBS by hPL led to improved osteogenic differentiation in vitro and more mineralized bone tissue in vivo.

The next objective of this PhD research was to enhance bone-forming properties in vivo by priming mandibular hPDCs with BMPs, seeding the cells on scaffolds and implant the constructs. Our work demonstrated that priming with BMP2 yields more pronounced effects regarding the upregulation of genes associated with the development of the skeletal system, when compared to BMP6. Among the tested scaffolds, CopiOs showed the most promising results in terms of ectopic in vivo bone formation. Furthermore, a rat model was developed for a critically-sized large bone defect in the mandible, and in this animal model the cell construct consisting of mandibular hPDCs, primed with BMP2, and then seeded on a CopiOs scaffold, was tested for its bone-regenerative abilities in a relevant environment.

Overall, this PhD research will contribute to the field of bone tissue engineering, with focus on cranio-maxillofacial bone repair, by exploring the potential of hPDCs, investigating different cell sources and culture conditions, and optimizing the use of BMPs and scaffolds for enhanced bone formation.

Date:1 Sep 2018 →  Today
Keywords:tissue engineering, bone
Disciplines:Laboratory medicine, Palliative care and end-of-life care, Regenerative medicine, Other basic sciences, Other health sciences, Nursing, Other paramedical sciences, Other translational sciences, Other medical and health sciences
Project type:PhD project