Oro-maxillofacial bone tissue engineering combining biomaterials, stem cells, and gene therapy

Improvements have been made in regenerative medicine, due to the development of tissue engineering and cellular therapy. Bone regeneration is an ambitious project, leading to many applications involving skull, maxillofacial, and orthopaedic surgery. Scaffolds, stem cells, and signals support bone tissue engineering. The scaffold physical and chemical properties promote cell invasion, guide their differentiation, and enable signal transmission. Scaffold may be inorganic or organic. Their conception was improved by the use of new techniques: self-assembled nanofibres, electrospinning, solution-phase separation, micropatterned hydrogels, bioprinting, and rapid prototyping. Cellular biology processes allow us to choose between embryonic stem cells or adult stem cells for regenerative medicine. Finally, communication between cells and their environment is essential; they use various signals to do so. The study of signals and their transmission led to the discovery and the use of Bone Morphogenetic Protein (BMP). The development of cellular therapy led to the emergence of a specific field: gene therapy. It relies on viral vectors, which include: retroviruses, adenoviruses and adeno-associated vectors (AAV). Non-viral vectors include plasmids and lipoplex. Some BMP genes have successfully been transfected. The ability to control transfected cells and the capacity to combine and transfect many genes involved in osseous healing will improve gene therapy.     

Tissue engineering

PURPOSE OF REVIEW: Regenerative medicine holds promise for the restoration of tissues and organs damaged by wear, trauma, neoplasm, or congenital deformity. Tissue engineering combines the disciplines of cell biology and biomedical engineering to effect the design and maturation of various tissues. Despite progress in some areas of tissue regeneration, there has not been significant translation to clinical practice. This article reviews the present understanding of and advances in regenerative medicine, as well as describing limitations in current techniques and areas that need further development. A discussion of the state of the art in the regeneration of skin, cartilage, bone, adipose tissue, and neural tissue is included. RECENT FINDINGS: Differences between extracorporeal and in-vitro tissue engineering are discussed, as well as tissue engineering principles, including the use of bioactive scaffolds, progenitor cells and stem cells, the need for cellular and tissue patterning, microcirculation development, and the use of external stimuli for differentiation. Much needs to be learned about progenitor cell biology, cell-cell interactions, cellular interactions with the extracellular matrix, and about the cues needed for differentiation of functional tissues. SUMMARY: The current limitations in regenerative medicine techniques and the gaps in current knowledge of cellular biology and tissue development represent significant research opportunities in tissue engineering.     

Cartilage and bone tissue engineering for reconstructive head and neck surgery

The loss of cartilage and bone because of congential defects, trauma and after tumor resection is a major clinical problem in head and neck surgery. The most prevalent methods of tissue repair are through autologous grafting or using implants. Tissue engineering applies the principles of engineering and life sciences in order to create bioartificial cartilage and bone. Most strategies for cartilage tissue engineering are based on resorbable biomaterials as temporary scaffolds for chondrocytes or precursor cells. Clinical application of tissue-engineered cartilage for reconstructive head and neck surgery as opposed to orthopedic applications has not been well established. While in orthopedic and trauma surgery engineered constructs or autologous chondrocytes are placed in the immunoprivileged region of joints, the subcutaneous transplant site in the head and neck can lead to strong inflammatory reactions and resorption of the bioartificial cartilage. Encapsulation of the engineered cartilage and modulation of the local immune response are potential strategies to overcome these limitations. In bone tissue engineering the combination of osteoconductive matrices, osteoinductive proteins such as bone morphogenetic proteins and osteogenic progenitor cells from the bone marrow or osteoblasts from bone biopsies offer a variety of tools for bone reconstruction in the craniofacial area. The utility of each technique is site dependent. Osteoconductive approaches are limited in that they merely create a favorable environment for bone formation, but do not play an active role in the recruitment of cells to the defect. Delivery of inductive signals from a scaffold can incite cells to migrate into a defect and control the progression of bone formation. Rapid osteoid matrix production in the defect site is best accomplished by using osteoblasts or progenitor cells.     

Clonal characterization of bone marrow derived stem cells and their application for bone regeneration

Tissue engineering allows the design of functionally active cells within supportive bio-scaffolds to promote the development of new tissues such as cartilage and bone for the restoration of pathologically altered tissues. However,all bone tissue engineering applications are limited by a shortage of stem cells. The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). BMSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate singlecell-derived colonies. These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, BMSCs have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues,including bone, cartilage, fat, tendon, muscle, and marrow stroma. Thus, BMSCs are an attractive cell source for tissue engineering approaches. However, BMSCs are not homogeneous and the quantity of stem cells decreases in the bone marrow in aged population. A sequential loss of lineage differentiation potential has been found in the mixed culture of bone marrow stromal cells due to a heterogenous population. Therefore, a number of studies have proposed that homogenous bone marrow stem cells can be generated from clonal culture of bone marrow cells and that BMSC clones have the greatest potential for the application of bone regeneration in vivo.     

Computer-aided three-dimensional reconstruction of the osseous and membranous labyrinths

Summary In order to obtain an adequate knowledge of the stereoscopic anatomy of the temporal bone, it is very useful to make three-dimensional reconstructions of the bone. By using the histological sections of a human temporal bone processed for routine evaluation, we have undertaken computer-aided reconstruction of the osseous and the membranous labyrinths, as well as the inner ear sensory organs. Reconstructions were done either separately or simultaneously. Owing to the semitransparent display function of the computer system, it was possible to observe the reconstructed membranous labyrinth through the simultaneously reconstructed osseous labyrinth, and the sensory organs through the membranous labyrinth. The results were satisfactory for understanding the spatial relationships among these structures. We also attempted computer-aided measurements of the reconstructed sensory organs and calculated the length of the organ of Corti and the angle between the saccular and the utricular maculae.     

Bone substitutes: Classification and concerns

Autograft is considered as the “gold standard” for bone reconstruction. It provides osteoinductive factors, osteogenic cells, and appropriate osteoconductive scaffold. Donor site morbidity is the main limitation of autograft. Donor disease transmission limits the use of allograft. Synthetic bone substitutes still lack osteoinductive or osteogenic properties. Composite bone substitutes combining synthetic scaffold and biochemical substances initiating proliferation and cell differentiation, and possibly osteogenesis. Bone substitutes and grafts intended for clinical use are listed.     

Scaffolds and biomaterials for tissue engineering: a review of clinical applications

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of organ function. This is achieved through implantation of cells/tissues grown outside the body or by stimulating cells to grow into an implanted matrix. In this short review, we discuss the use of biomaterials, in the form of scaffolds, for tissue engineering and review clinical applications to otorhinolaryngology-head and neck surgery.     

Tooth regeneration: a revolution in stomatology and evolution in regenerative medicine

A tooth is a complex biological organ and consists of multiple tissues including the enamel, dentin, cementum and pulp. Tooth loss is the most common organ failure. Can a tooth be regenerated? Can adult stem cells be orchestrated to regenerate tooth structures such as the enamel, dentin, cementum and dental pulp, or even an entire tooth? If not, what are the therapeutically viable sources of stem cells for tooth regeneration? Do stem cells necessarily need to be taken out of the body, and manipulated ex vivo before they are transplanted for tooth regeneration? How can regenerated teeth be economically competitive with dental implants? Would it be possible to make regenerated teeth affordable by a large segment of the population worldwide? This review article explores existing and visionary approaches that address some of the above-mentioned questions. Tooth regeneration represents a revolution in stomatology as a shift in the paradigm from repair to regeneration: repair is by metal or artificial materials whereas regeneration is by biological restoration. Tooth regeneration is an extension of the concepts in the broad field of regenerative medicine to restore a tissue defect to its original form and function by biological substitutes.     

Regenerative medicine in the fields of otolaryngology; head and neck regions

Because of the limitations of the present medicine, regenerative medicine has greatly been hoped. Regenerative medicine is an exciting emerging branch of medicine in which cell and tissue based therapies are applied to the treatments of disease. This has been supported by the development of tissue engineering that was a complex of medicine and engineering. Tissue engineering applies the principles and methods of engineering, material science, and cell and molecular biology toward the development of viable substitutes which restore, maintain, or improve the function of human tissues. According to the doctrine of tissue engineering, tissues and organs can be regenerated by manipulating three elements: cells, scaffolds and regulation factors. Understanding and manipulating the complex relationship among these elements, however, represents the great challenge for researches and doctors who engage in regenerative medicine. On the other hand, clinical applications of it have not made progress yet except limited tissues and organs. Especially, few clinical studies have reported in the fields of otolaryngology, head and neck regions. In our research group, clinical applications in these fields based on the new concept of in situ tissue engineering have been started from 2002 after approval of the ethical committee. Tissues and/or organs for clinical applications at present are trachea, cricoid cartilage, mastoid air cells, and, peripheral nerves including facial nerve, recurrent laryngeal nerve, chorda tympani nerve and so on. This article describes the researches and clinical studies at present and in the near future of regenerative medicine in the fields of otolaryngology, head and neck regions.     

Tissue integration of the collagen-hydroxylapatite implant: histological examination in canine bone and surrounding tissues

Summary Using the dog as an animal model, we have tested an implant material composed of purified fibrillar collagen (PFC) and particulate hydroxylapatite (HA) in the mandible and in surrounding tissues. Bone and tissue samples were taken at 2, 4 and 6 months for histological study. After 2 months, the PFC was replaced by fibroconnective host tissues. After 4 months, some small areas of ossification were observed around the HA particles. After 6 months, the fibro-connective tissue was replaced by neo-formed bone in the mandible. PFC was found to increase the interfaces between the HA particles and the host tissues, permitting HA integration into the bone. The PFC/HA implant was also molded when moistened by blood or saline solution and then became mis-shapen by local pressures exerted. These findings show that the implant should preferably be reserved for the restoration of bones not subjected to significant forces or local stresses. Offprint requests to: M. Remacle     

Tissue engineering: the current status of this futuristic modality in head neck reconstruction

PURPOSE OF REVIEW: To define tissue engineering and describe the components that are involved in engineering tissue. To provide examples of the most recent developments in tissue engineering as they apply to the otolaryngologist. To provide a general overview of selected exciting advances outside the field of general otolaryngology but of general interest to the reconstructive community. RECENT FINDINGS: Tissue engineering is a rapidly evolving field that can be defined as the regeneration of new tissues through the use of biologic mediators and matricis. The three components required are scaffolds, signaling molecules, and cells. A significant amount of work has been done to generate bone, cartilage, cornea, and vascular grafts. Tissue engineered bone is the only tissue type that has been evaluated in Phase III clinical trials and is in routine clinical use. Engineered bone from BMP-2 and BMP-7 is used in orthopedics for lumbar fusions and long bone nonunions. Clinical application of bone tissue engineering in the head and neck is limited to case reports. There has been success in tissue engineering with cartilage for the nose and ear in immune incompetent animal models but there has been difficulty with generating scaffolds that do not incite an immune reaction in an immune competent model. The most advanced organ engineered is a bladder that has been successful in a canine model. SUMMARY: Tissue engineering holds the promise of "off the shelf" parts for reconstruction of tissues and organs. The widest clinical application is with bone in orthopedics. Applications in the head and neck are limited, at present, due to the immunogenicity of the scaffolds and the challenging wounds encountered in both cancer and cosmetic patients.     

Electron microscope study of bone lesion in cholesteatoma otitis

Summary Biopsy specimens were obtained at ear surgery of patients with cholesteatoma otitis and were studied by electron microscopy. The osseous tissue decalcified in EDTA demonstrated degradation of collagen matrix as well as extensive degeneration of bone cells. The surface of the eroded bone was found to be in direct contact with the subepithelial connective tissue or the granulation, but not with the keratinizing squamous epithelium of matrix. These results indicate that the bone destruction by cholesteatoma is of non-osteoclastic type and lend support to the view that the keratinizing squamous epithelium do not participate in bone destruction. On the ultrastructural basis, the inflammatory process in the soft tissue adjacent to bone and extensive degeneration of bone cells are considered as cardinal factors for bone destruction in this disease.     

Tissue-engineered bone formation with gene transfer and mesenchymal stem cells in a minimally invasive technique

BACKGROUND: The objective of this study was to use a chitosan-alginate gel to implant bone marrow-derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote bone formation by the simultaneously transferred osteogenic protein (OP)-1 (bone morphogenic protein-7) gene. METHOD AND RESULTS: The complex of polyethylenimine/luciferase plasmid DNA embedded in the gel was able to transfect HEK 293 cells on a culture dish or co-encapsulated in the gel. When injected into the subcutaneous space of mice, luciferase expression was two to three orders of magnitude increased above the background. To examine the efficacy of gene-, cell-, and combined gene- and cell-encapsulated gels in tissue generation, samples were injected into the subcutaneous space of 6-week-old athymic nude mice, and the OP-1 plasmid was studied. At 8 weeks after the injection, the gels only maintained their volumetric shape when human mesenchymal stem cells (hMSCs) were encapsulated, but otherwise the gels were partially dissolved. Transgene expression of OP-1 was clearly detected in the samples after 4 weeks but not after 8 weeks. Type II collagen was detected in all the gels containing the OP-1 plasmid, with or without hMSCs. The samples with the combination of OP-1 DNA and hMSCs revealed strong type II collagen expression as well as osteoid foci. CONCLUSION: These results suggest that combined gene and hMSC delivery within a chitosan-alginate gel could be an interesting approach for tissue engineering.     

Regeneration of the vocal fold using autologous mesenchymal stem cells

The aim of this study was to regenerate the injured vocal fold by means of selective cultured autologous mesenchymal stem cells (MSCs). Eight adult beagle dogs were used for this experiment. Selective incubation of MSCs from bone marrow was done. These MSCs were submitted to 3-dimensional incubation in 1% hydrochloric acid atelocollagen. Three-dimensional incubated MSCs were injected into the left vocal fold, and atelocollagen only was injected into the right vocal fold of the same dog as a control. Four days after injection, the posterior parts of the vocal folds were incised. The regeneration of the vocal fold was estimated by morphological and histologic evaluations. Our results showed that 3-dimensional incubated MSCs were useful in the regeneration of the injured vocal fold. This study shows that damaged tissues such as an injured vocal fold would be able to be regenerated by tissue engineering.     

Computed tomography demonstrates abnormalities of contralateral ear in subjects with unilateral sensorineural hearing loss

Purpose

Prior studies have associated gross inner ear abnormalities with pediatric sensorineural hearing loss (SNHL) using computed tomography (CT). No studies to date have specifically investigated morphologic inner ear abnormalities involving the contralateral unaffected ear in patients with unilateral SNHL. The purpose of this study is to evaluate contralateral inner ear structures of subjects with unilateral SNHL but no grossly abnormal findings on CT.

Materials and methods

IRB-approved retrospective analysis of pediatric temporal bone CT scans. 97 temporal bone CT scans, previously interpreted as “normal” based upon previously accepted guidelines by board certified neuroradiologists, were assessed using 12 measurements of the semicircular canals, cochlea and vestibule. The control-group consisted of 72 “normal” temporal bone CTs with underlying SNHL in the subject excluded. The study-group consisted of 25 normal-hearing contralateral temporal bones in subjects with unilateral SNHL. Multivariate analysis of covariance (MANCOVA) was then conducted to evaluate for differences between the study and control group.

Results

Cochlea basal turn lumen width was significantly greater in magnitude and central lucency of the lateral semicircular canal bony island was significantly lower in density for audiometrically normal ears of subjects with unilateral SNHL compared to controls.

Conclusion

Abnormalities of the inner ear were present in the contralateral audiometrically normal ears of subjects with unilateral SNHL. These data suggest that patients with unilateral SNHL may have a more pervasive disease process that results in abnormalities of both ears. The findings of a cochlea basal turn lumen width disparity >5% from “normal” and/or a lateral semicircular canal bony island central lucency disparity of >5% from “normal” may indicate inherent risk to the contralateral unaffected ear in pediatric patients with unilateral sensorineural hearing loss.     

Distinct spontaneous shrinkage of a sporadic vestibular schwannoma

We present a case with outspoken spontaneous vestibular schwannoma shrinkage and review the related literature. The patient was initially diagnosed with a left-sided, intrameatal vestibular schwannoma, which subsequently grew into the cerebello-pontine angle (CPA), followed by total shrinkage of the CPA component without any intervention over a 12-year observation period. The literature on spontaneous tumor shrinkage was retrieved by searching the subject terms “vestibular schwannoma, conservative management” in PubMed/MEDLINE database, without a time limit. Of the published data, the articles on “shrinkage” or “negative growth” or “regression” or “involution” of the tumor were selected, and the contents on the rate, extent and mechanism of spontaneous tumor shrinkage were extracted and reviewed. The reported rate of spontaneous shrinkage of vestibular schwannoma is 5–10% of patients managed conservatively. Extreme shrinkage of the tumor may occur spontaneously.     

Exploration of the relationships among medical health history variables and aspiration

Objectives

(1) Determine the relationships among a family's specific answers to health history questions and their child's risk for aspiration as determined by a swallow study. (2) Identify key factors which may predict a child's risk for aspiration.

Methods

Answers to questions and results of Functional Endoscopic Evaluation of Swallow (FEES) and/or Video Swallow Study (VSS) for a sample of 198 children were collected. Subjects were categorized into groups: “aspiration”, “penetration” or “no aspiration or penetration”. Logistic regression modeling was used to determine the contribution of certain characteristics to the odds of aspiration. A model for predicting aspiration or penetration based on those characteristics was assessed. The sensitivity and specificity of the model in predicting aspiration and penetration was determined.

Results

One hundred ninety eight subjects had a FEES or VSS. Of these, 18% (n = 36) aspirated and 21% (n = 42) were found to have penetration. Many of the factors identified in the health history were found to be significantly associated with aspiration or penetration. The variables “demonstrated aspiration” (this included parent or caregiver seeing food in the tracheotomy tube or aspiration noted on a previous FEES or VSS) (p = 0.02), “hypotonia” (p = 0.02) and “tracheotomy” (p = 0.001) were most predictive of aspiration. History of tracheotomy was found to have an inverse relationship with aspiration. “gastroesophageal reflux” (GER) (p = 0.0007) was most significantly associated with penetration, followed by “prematurity” (p = 0.03) and “developmental delays” (p = 0.04). Based on the prediction model, the probabilities for a child with a history of combinations of the above variables to have aspiration or penetration were calculated.

Conclusions

Significant relationships exist between aspiration or penetration and the family's answers about their child's medical history. Practitioners should consider a swallow assessment whenever a child has a history which includes variables with a strong association with aspiration or penetration.     

Effects of FGF-2 and OP-1 in vitro on donor source cartilage for auricular reconstruction tissue engineering

Objective

Microtia is a congenital partial or total loss of the external ear with current treatment approaches involving autologous construction from costal cartilage. Alternatively, tissue engineering provides possible use of normal or microtia auricular chondrocytes harvested from patients. This study investigated effects in vitro of basic fibroblast growth factor (FGF-2) and osteogenic protein 1 (OP-1) on human pediatric normal and microtia auricular chondrocytes and their potential proliferation and differentiation for cellular expansion. A working hypothesis was that FGF-2 promotes proliferation and OP-1 maintains an auricular phenotype of these cells.

Methods

Two patients, one undergoing otoplasty and one an ear construction, yielded normal and microtia auricular chondrocytes, respectively. The two donor sets of isolated chondrocytes were equally divided into four experimental cell groups. These were controls without added growth factors and cells supplemented with FGF-2, OP-1 or FGF-2/OP-1 combined. Cells were cultured 3, 5, 7, and 10 days (3 replicates/time point), counted and assayed by RT-qPCR to determine elastin and types II and III collagen gene expression.

Results

Compared to control counterparts, normal and microtia chondrocytes with OP-1 alone were similar in numbers and varied in elastin and types II and III collagen expression over all culture times. Compared to respective controls and chondrocyte groups with OP-1 alone, normal and microtia cell groups with FGF-2 had statistically significant (p < 0.05) enhanced proliferation and statistically significant (p < 0.05) decreased elastin and types II and III collagen expression over 10 days of culture.

Conclusions

FGF-2 effects on normal and microtia chondrocytes support its use for increasing cell numbers while OP-1 maintains a chondrocyte phenotype, otherwise marked by increasing type III collagen expression and cellular dedifferentiation to fibroblasts in culture.     

Some morphological details of the endochondral layer of labyrinthine bone

Summary The endochondral layer of labyrinthine bone of dog (Canis f. intermedius Woldrich) is examined by means of light and transmission electron microscopy.The osteocytes of the fine-fibred, alamellar embryonic skein bone (Meyer 1927) are surrounded by a fibre-less matrix. They correspond to osteocytes in long bones. Some of them are producing microfibrils according to formation of preosseous tissue which is called intraosseous osteogenesis (Knese 1970). Others are characterized by signs of micropetrosis or indicate osteocytic osteolysis. In the perivascular mesenchym of some vessels giant cells are visible which according to their ultrastructural characteristics are identified as osteoclasts.The occurrence of an embryonic type of bone in endochondral layer of otic capsule throughout life is discussed considering oxygenation via perivascular canalizition as well as biomechanical aspects.Dedicated to Prof. Dr. B. Schloßhauer, Bremen, on the occasion of his 60th birthday