Читать книгу Bone and Soft Tissue Augmentation in Implantology - Группа авторов - Страница 16

Bone regeneration is another important aspect of bone biology. Bone regeneration works perfectly in the sense that no scar tissue is formed, which contrasts with the classical skin wound healing in adults, where the defect is left with a matrix rich in collagen but poor in cells. This is summarized in excellent reviews on bone regeneration, particularly in fracture healing^30,42 and wound healing.^90,113,143 Both events start with the formation of a blood clot, where the coagulation cascade of proteases culminates in the formation of thrombin, which cleaves fibrinogen. The fibrin itself assembles into a transient extracellular matrix, where platelets are activated and form aggregates, together with erythrocytes. Growth factors and other molecules are released, attracting neutrophils into the blood clot to clean the defect site. Macrophages appear later in the blood clot. To make space for the granulation tissue, which is characterized by the sprouting of blood capillaries into the new tissue and the concomitant appearance of fibroblastic cells, fibrinolysis is initiated. The invading cells release activators for plasminogen being stored in the blood clot – it is plasmin that cleaves the fibrin matrix. Interestingly, mouse models lacking fibrinogen allow bone regeneration,¹⁴¹ while those lacking plasminogen show impaired bone regeneration.⁶⁴ These findings highlight the importance of fibrinolysis over the formation of the fibrin matrix.

Mouse models have also helped in the understanding of the importance of macrophages in bone regeneration, as they were shown to be in wound healing, early on. The depletion of macrophages and the genetic modification of the cells to erase their activity culminate in impaired bone regeneration, including intramembranous ossification, which is the more relevant path in regenerative dentistry compared with the endochondral ossification that is typically observed in fracture healing.^95,135 However, the role of macrophages is not restricted to a defect situation. For example, macrophages form a canopy structure over mature osteoblasts during bone remodeling, suggesting that they interact via juxtacrine and a paracrine mechanism that remains to be fully elucidated.²⁵ The clinical implication of this fundamental principle in regenerative dentistry is unclear, but it opens a wide arena for research that may involve biomaterials. Mouse models have also provided evidence that at least a transient inflammation is required for bone regeneration, as, for example, the knockout of TNFα^24,48 and COX-2¹⁴² caused impaired bone regeneration. Moreover, in bones lacking bone morphogenetic protein 2 (BMP-2), the earliest steps of fracture healing seem to be blocked,¹²⁵ and it is possible that the local inflammation controls the expression of BMP-2, at least in vitro.⁴⁶ To what extent macrophages are involved in the inflammation required for bone regeneration has not yet been investigated. Also, here, the clinical relevance of these observations should be interpreted with care. For example, painkillers should not be a great concern in regenerative dentistry as they do not completely block cyclooxygenases and are only used temporarily.⁴⁹ Bone regeneration is not influenced or jeopardized when inhibitors of TNFα are used,¹²² as in a situation of chronic inflammation, including rheumatoid arthritis and colitis ulcers. Thus, findings from the extreme situation of a gene knockout or enhanced expression in mouse models should be interpreted carefully in the clinical context.

Mouse models also support the role of BMP-2 during bone regeneration.¹²⁵ Molecular screening approaches have revealed a long list of growth and differentiation factors that are differentially expressed during bone regeneration, in particular fracture healing, that play a major role in bone formation.⁵⁴ For example, BMP-4¹²⁶ and BMP-7¹²⁷ have no effect on fracture healing, but Wnt signaling is crucial for bone regeneration, based on observing with a sclerostin antibody and sclerostin knockout models.⁴ The Hedgehog signaling pathway also plays a critical role in osteoblasts during fracture repair.⁶ While it is obviously the orchestrated interplay of a large spectrum of local and systemic signals that drives osteoblastogenesis, and thus bone regeneration, there are growth factors such as BMP-2 that are not only supportive but also essential for proper bone regeneration, and thus likely also for graft consolidation. However, considering the complex interplay of immune cells, endothelial cells, osteocytes, and osteoclasts in controlling bone formation, many molecular mechanisms remain to be discovered.

Histology has provided insights into the defect sites, showing that osteoclasts are already active a few days after the injury, and that bone formation by osteoblasts is clearly visible 10 days after implant insertion in a pig model.¹³¹ The new bone grows fairly rapidly, at approximately 10 µm per day, and sprouts into the defect area. Then, lamellar bone is formed on the surface of the woven bone, which overall is independent of osteoclasts and is thus strictly in an anabolic phase until bone remodeling is initiated. Finally, the woven bone and the primary lamellar bone are replaced by secondary lamellar bone, which is the final stage of bone regeneration, and bone remolding takes over. What histology convincingly demonstrates is that the new bone grows into an area rich in blood vessels, but without touching them.¹³¹ Considering the three choices of osteoblasts – to become an osteocyte, to become a lining cell, or to die – a supply of new osteoblasts to drive bone regeneration seems mandatory. The close proximity of osteoblasts has always pointed toward blood vessels as the source of the mesenchymal progenitor cells, but evidence was scarce. Today, advanced mouse models have supported this hypothesis, e.g. by showing that only a certain type of endothelial cells (H-type) is associated with osteogenic precursors, which resemble pericytes but are perhaps a distinct population.^78,134 Blood vessels in the growing long bones are rich in osteogenic precursors, and are predetermined as they express the differentiating marker osterix.⁷⁸ Blood vessels in the bone marrow, however, do not carry this cell population. It is reasonable to suggest, under the premise that (to some extent) bone regeneration recaptures bone development, that these osteogenic blood vessels also sprout into the defects after implant insertion or bone augmentation. Moreover, Prx1-Cre mouse models support the role of the periosteum as a rich source of osteogenic cells. These cells can efficiently contribute to cartilage and bone formation upon injury.⁴¹ This knowledge now has to be translated into higher animals and its clinical relevance determined.

Fig 1-6 Dental implant after 5 days of osseointegration in a pig jaw, from a study by Vasak et al.¹³¹ Five days after implant placement, close to the implant surface, the bone is fragmented, squeezed, and heat damaged (dark pink). Osteocytes in the vicinity are dead and dying. Osteoclasts (white asterisks) are digging bone channels, sprouting from existing bone canals (osteons) to reach and resorb the damaged bone. They are about to reach the most damaged bone close to the implant and will soon remove it.

If the overall hypothesis is correct, the formation of this subtype of endothelial cells carrying the osteogenic cells is essential for bone regeneration, and thus also for regenerative dentistry. However, since the pioneer findings with parabiosis experiments,²² there is good evidence that blood vessels provide the progenitors of osteoclasts. Here, the bone marrow is irradiated and thus osteoclastogenesis is impaired; however, it is regained when the circulation is connected to a vital mouse, so that osteoclast progenitors have to be carried via the bloodstream. Taken together, the blood vessels are key for osteoblastogenesis and osteoclastogenesis – and consequently also for bone regeneration.