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21 Bone andCartilage Tissue Engineering andRegenerative Medicine inCraniofacial Surgery
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• Iliac crest [75, 76]
• Fibula [75, 77, 78]
• Scapula [79–81]
The major advantage over non-vascularized bone grafts is that vascularization allows ade­quate nutrient supply even in very large grafts. Due to their own permanent vascularization and the preserved vitality of the vascularized bone graft, they show in the recipient site a faster healing, a more pronounced adaptation to the new mechanical conditions, an earlier onset and higher mechanical loading capacity, a higher survival and healing rate in the case of bearing that is incapable of replacement and weak (infection, chemotherapy, radiotherapy, poor vascularization), and a neovascularization potency on the surrounding tissue [82]. However, in contrast to non-vascularized grafts, the signicantly greater donor-site morbidity must be accepted.
Flap Prelamination andPrefabrication
Flap prefabrication forms a transition from con­ventional reconstructive surgery to tissue engi­neering [83]. Following the exciting fabrication of a human ear on a mouse back by Cao and Vacanti [84], this novel medical technology is also becoming widely recognized by the public. Prefabrication involves the insertion of a new vascular pedicle into the ap to be transplanted, so that the ap graft can be transplanted with the added vessels as the main supply after a period of neovascularization. Both techniques allow the shaping of individual aps consisting of several tissues, which can be used especially for complex reconstructions in the area of the face, nose, or ears [85].
Prelamination of a ap plasty involves the introduction of additional tissue into the graft to be transplanted into a microvascularly anasto­mosed musculocutaneous ap [70, 83, 86–91].
Another promising approach is vascular induction. Bone chips have already been inserted into muscle tissue, resulting in a vascularized
bone graft [92]. Future approaches could attempt to create complex vascularized tissues.
Even if the research is not yet fully completed, there are already initial feasibility studies that show promise. It has already been possible to transplant a customized vascularized bone graft into a human [93]. However, further research efforts need to clarify more details before this therapeutic procedure enters clinical routine.
Extracorporeal Strategies
In tissue engineering, cells are usually taken from the organism of a donor and cultivated in vitro in the laboratory. These can then be implanted into usually the same organism and thus preserve or restore a tissue function. Sufcient tissue engineering requires the fol­lowing main components [1]:
– Bone or cartilage cells – Scaffold (two- or three-dimensional) – Growth factors – Culture medium
Thus, biomaterials can be assembled in biore­actors to form a complex three-dimensional con­struct invitro (Figs.21.7 and 21.8). The scaffolds should degrade themselves in the process. However, new research approaches are attempt­ing to generate scaffold-free three-dimensional microtissue constructs (Figs. 21.9 and 21.10). Growth factors also appear to play an important role in the extracorporeal strategy, as described in the chapter Biological Stimuli. Once again, it is evident that the interaction of all factors with and among each other only leads to the successful construction of tissue substitutes by simple cells. The sequence can last from a few seconds to weeks, with sizes ranging from 1μm to 10cm. The step from experimental studies to clinical application is already taking place [19].
The human bone as an organ of the skeletal system consists of many very different cells, all of which serve the bone in its entirety with their specic function. These include:
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Cartilage engineering
Mineralisaon of stem cell microsphere cultures. USSC: Ubbilicalstemcells; ES: embryonicstemcells
s
Microsphere related bone engineering–cultureproducts
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defect
Cartilage healing after
defectfilling
Fig. 21.7 Steps of cartilage engineering (explantation and culture control)
V. Kerkfeld et al.
Defect histologyFullsize cartilage
Control USCC
ESC USCC micromas
Fig. 21.8 Steps of cartilage engineering (implantation and clinical control)
postoperavely
Microsphere relatedboneengineering –
Techniques to investigatebone tissue
TEM
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ectopicinsitu bone formation
USCC
ESC
Control
micromass
USCC
ESC
Control
289
USCC
micromass
USCC
1 month
postoperavely
Fig. 21.9 Microsphere-based bone tissue engineering during culture
4 month
engineeringapproaches
-Microanalysis-
Protein analysis
Mineralisation
Mineral structure
SEM
High resolution (nm)
collagen analysis
Electron diffraction
analysis (EDA)
Fig. 21.10 Microsphere-based bone tissue engineering after in situ implantation
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– Vascular cells – Bone marrow cells – Osteocytes and their precursor cells – Chondrocytes and their precursor cells – Osteoclasts
In vitro, on the other hand, even a limited number of cell types is sufcient to produce extracorporeal bone tissue. Different donors, which are in different stages of scientic devel­opment, serve as cell sources (Table21.1).
In this context, the cells from the individual donor sources (e.g., autologous or xenogeneic) can be further differentiated according to their degree of determination into embryonic and adult stem cells as well as into determined cells. Some successful approaches with both mature and pro­genitor cells have already been implemented for bone tissue engineering [94]. In addition to this research, immortalized cells have also been used to further evaluate cell behavior invitro.
Skeletal tissue stem cell is a hypothetical con­cept (for review, see Triftt [95]). Stem cells can be divided into embryonic and adult. Embryonic stem cells are found in blastocysts and were suc­cessfully grown more than 40 years ago [96]. Those stem cells that give rise to bone structures are referred to in a scientically inconsistent manner, with the term mesenchymal stem cell increasingly gaining acceptance [97–99]. Stem cells can replicate indenitely without reaching a degree of differentiation. Therefore, their daugh­ter cells can subsequently form into a wide vari­ety of tissues. Thus, in addition to bone and cartilage, tendons, muscles, fat, and bone marrow stroma are also formed. Due to this developmen­tal diversity, stem cells are of great importance
Table 21.1 Principal cell sources used in tissue engi­neering studies. Adopted from Meyer, Meyer [1]
Preclinical
Experimental studies Autologous Autologous Autologous Allogenic Allogenic Xenogeneic Xenogeneic Immortalized Non-transformed
clonal Sarcoma
studies
Clinical studies
for tissue engineering (for review, see Saeedi, Halabian [100]). Marrow stem cells (MSCs) are present in fetal tissue and adult bone marrow [99]. The bone marrow serves as the primary res­ervoir for MSCs. However, they could be found in many other specialized tissues:
– Periosteum [101] – Liver [102] – Blood [102]
Clinically, this can be exploited by covering cartilage defects with periosteum, for example. The existence of MSCs in adult peripheral blood has not yet been clearly established. What is clear, however, is that when present, it is only an extremely small amount of MSCs in peripheral blood [103].
In clinical application, however, the use of adult stem cells causes some problems. First and foremost is the fact that it is difcult to collect a sufcient number of stem cells. For this reason, the use of fetal stem cells in particular has become more and more established [1]. Fetal tissue secretes an environment that signicantly pro­motes growth, e.g., through growth factors. Stem cells from fetal bone marrow in particular have the further advantage that they are signicantly less immunogenic. Therefore, signicantly less attention needs to be paid to tissue typing, which is important when using adult stem cells. In addi­tion, fetal stem cells are signicantly more primi­tive in terms of their degree of differentiation. Thus, they still offer a versatile differentiation potential and can thus develop into a wide variety of cells from the mesenchymal cell lineage. In summary, fetal stem cells thus offer very good conditions for tissue engineering. However, these properties also have negative implications. For example, it is not yet possible to specically con­trol the differentiation of embryonic stem cells. Moreover, it has already been shown in animal experiments that embryonic stem cells carry a degeneration potential after transplantation [104]. Similarly, immunological incompatibility reactions may occur after transplantation [105]. However, in addition to these biological limita­tions, other hurdles arise in clinical applications.
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For example, legal and ethical issues must be considered, sometimes even separately in differ­ent areas of application.
Furthermore, both the propagation and differ­entiation of stem cells in the clinical setting pres­ent major difculties. Especially for adult stem cells, it remains difcult to generate a sufcient number of cells invitro in the clinically required time [1]. Unfortunately, the application has not yet reached the stage where functional, mature osteocytes could be generated that would allow physiological bone mineralization invitro [106,
107]. Therefore, much research remains to be
done for nal application. There is great anticipa­tion of possible gene therapy using gene insertion [108, 109].
Determined Bone Cells
Determined osteoblast-like cells are widely used in the scientic and clinical application of bone tissue engineering. Osteoblasts in any precursor stage can be harvested and cultured. In addition, genetic modications to the cells are possible, but so far these are only underway in experimen­tal studies. Each cell source has its own specic advantages and disadvantages.
Autologous, determined osteoblast-like cells are currently considered the most important cell source in the eld of bone tissue engineering [110]. On the one hand, this is since they are not subject to any legal or moral restrictions. On the other hand, no immunological reactions are to be expected because the cells are endogenous. However, the supply of autologous bone material is limited, so that large defects sometimes cannot be completely lled. The previously held scien­tic opinion that adult stem cells are subject to reduced replication and viability has now been refuted in recent studies. For the cultivation of osteoblasts, different maturation stages of pro­genitor cells are used. The progenitor cells differ in many ways, e.g., by different gene expression, resulting in a specic prole of transcription fac­tors, cell cycle regulatory proteins, adhesion mol­ecules, or even matrix proteins (for review, see Yamaguchi, Komori [94]).
Bone cell populations can be taken from dif-
ferent collection sites. These include:
• Corticalis
• Cancellous bone
• Bone marrow
• Periosteum
The respective cells can subsequently be iso­lated enzymatically or mechanically [111]. They may differentiate into broblastic, osteogenic, or reticular cells [112–115]. It remains unclear whether either cortical or cancellous bone creates a better condition for cell cultures [116–118]. However, it has already been shown that the cul­tivation of particles is superior to the cultivation of bone chips [119, 120]. It is assumed that the size of the surface is relevant. The larger the sur­face, the more cells are exposed and can be culti­vated. However, there is also controversial scientic evidence on the subject. In an experi­mental study, bone chips from trabecular bone yielded signicantly more released living cells than from cortical bone [89]. Furthermore, it was shown that after processing cancellous bone with a bone mill towards bone particles, it did not yield more but, conversely, fewer osteoblast-like cells. This suggests that reduced particle size may be detrimental to cell seeding, contrary to previous opinion. In contrast, enzymatic diges­tion of bone was able to provide excellent results. First, Ecarot-Charrier and Glorieux [121] suc­ceeded in exposing and subsequently isolating osteoblasts from mouse calvaria by enzymatic process. The osteoblasts thus obtained retained their properties in culture after the exposure pro­cedure, so that this method became the gold stan­dard for invitro cell harvesting.
After isolation of osteoblast-like cells, many factors can inuence the expression and differ­entiation of the phenotype. The culture medium, cultivation time, and surrounding substances are particularly relevant for this. These substances include ascorbic acid, β-glycerophosphate, as well as dexamethasone. β-Glycerophosphate physiologically serves as a phosphate donor and protein phosphatase inhibitor. Thus, it supports the development of scaffolds in bone tissue
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Techniquesto investigate bone tissue
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engineering and is also used to drive osteogenic differentiation of bone marrow stem cells in vitro. It enables phenotypic matrix matura­tion and promotes mineral formation in osteo­blast-like cell cultures [1]. Dexamethasone has an ambivalent effect on cultivation. On the one hand, dexamethasone promotes differentiation but, on the other hand, inhibits proliferation [122–124]. It is advisable to establish a stan­dardized cultivation protocol to standardize the product grown exvivo.
Physiologically, bone formation and heal­ing of bone fractures proceed via a cartilagi­nous intermediate. Therefore, the research approach that chondrocytes could improve bone regeneration became established [113]. However, further research efforts showed that although chondrocytes could generate histo­logically similar cartilage, it did not initiate endochondral ossification. It is believed that mature chondrocytes inhibit angiogenesis, thus preventing the endochondral cascade [106]. More recent approaches attempt to alter the chondrocyte phenotype so that hypertrophic chondrocytes can be grown. However, the methods are not yet mature [125, 126].
Evaluation ofEngineering Success
Bone tissue engineering attempts to restore large bone defects [127, 128]. Animal models can be used to attempt to adequately reect the clinical situation of human bone healing. To evaluate the success of exvivo-fabricated bone tissue substi­tutes, several assessments must be made on a his­tological and microanalytical level (Figs.21.11 and 21.12) (Table21.2).
The evaluation of different bone tissue engi­neering approaches is strongly dependent on the type of experimental model [129].
To date, only a basic understanding of the complex processes involved in bone tissue engi­neering is available. In addition, the processes in the various surgical elds of activity differ sig­nicantly. In oral and maxillofacial surgery, good bony regeneration is often seen after sinus lift. This often occurs even without the use of bioma­terials. It seems that the crucial inductive step is the lifting of the maxillary sinus mucosa [1]. To investigate the osteoinductive potential, hybrid materials are used invivo. The hybrid material is implanted ectopically, e.g., in muscle pockets. Subsequently, the critical defect size must be
Tissue analysis
Histology
Fig. 21.11 Approaches to control the success on a histological level (e.g., in clinical studies, but also used to investi­gate extracorporeally generated tissue constructs)
engineering approaches
-Histology-
In situ tracking Cell/Protein synthesis
Histology
Immunolabeling
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Bone repair by
autologous cells
293
Augmentaon of local host cells
Membranes
Guided bone
regeneraon
Fig. 21.12 Approaches to control the success on a microanalytical level (mainly used to investigate extracorporeally generated tissue constructs but can also be applied in clinical studies)
Table 21.2 Animal experimental and clinical evaluation methods. Adopted from Meyer, Meyer [1]
Method Determination Clinical Form and function X-ray Hard tissue structure (overview) Microradiography Hard tissue structure (details) CT Hard and soft tissue structure
μCT
Densitometry Hard and soft tissue structure Serum analysis Mineral content Urine analysis Bone formation and resorption
Histology (in vitro)
Biophysical smuli
Distracon
Ultrasound
Electromagnec
fields
(overview) Hard and soft tissue structure (details)
marker, hormones, cytokines Tissue structure
Biological smuli
Cytokines
Cell-based grafting approaches are currently very popular among clinical users to support and enhance bone tissue healing. This is partly due to the legal situation that allows the handling of autologous cells.
Conclusion
Tissue engineering and regenerative medicine offer craniofacial surgery far-reaching possibilities to overcome existing limitations in patient care. Especially in the eld of osseous and cartilaginous tissue engineering, considerable scientic prog­ress has been made. However, heterogeneity in
Transplantaon of cells
non-vascularized
cells
cancellous bone
corcal bone
bulk bone
vascularized
pedicled
free
study conditions does not allow for uniformly
determined by means of an invivo defect model. The choice of the test animals and the defect site is crucial, as it has already been shown that there are signicant differences in bone regeneration depending on the degree of maturity of the ani­mals [130, 131] and the mechanical bone load.
valid conclusions for clinical application. Scientic investigation of this highly exciting topic is pro­gressing with great strides. Large-scale, system­atic studies may in the future produce uniform recommendations on the most effective cell types, scaffolds, growth factors, and culture media.
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