An overview of decellularisation techniques of native tissues and tissue engineered products for bone, ligament and tendon regeneration.

Decellularised tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications. Because of the complexity of each tissue (size, porosity, extracellular matrix (ECM) composition etc.), there is no standardised protocol and the decellularisation methods vary widely, thus leading to heterogeneous outcomes. Physical, chemical, and enzymatic methods have been developed and optimised for each specific application and this review describes the most common strategies utilised to achieve decellularisation of soft and hard tissues. While removal of the DNA is the primary goal of decellularisation, it is generally achieved at the expense of ECM preservation due to the harsh chemical or enzymatic processing conditions. As denaturation of the native ECM has been associated with undesired host responses, decellularisation conditions aimed at effectively achieving simultaneous DNA removal and minimal ECM damage will be highlighted. Additionally, the utilisation of decellularised matrices in regenerative medicine is explored, as are the most recent strategies implemented to circumvent challenges in this field. In summary, this review focusses on the latest advancements and future perspectives in the utilisation of natural ECM for the decoration of synthetic porous scaffolds.

Pro-osteogenic properties of hydrophilic and hydrophobic titanium surfaces: Crosstalk between signalling pathways in in vivo models.

BACKGROUND: It is now generally accepted that the response to a particular signal, such as the surgical trauma following implant placement, is not the result of a single linear signalling pathway, but rather reflects pathway integration, which can occur at multiple levels. Although it is well documented that both SLA and SLActive surfaces are able to promote bone formation and osseointegration, it is still unclear which are the key signalling pathways involved and how surface hydrophilicity/hydrophobicity might affect pathway integration. OBJECTIVE: To combine gene and protein data from in vivo studies applying titanium hydrophobic (Sandblasting, Large-grit, Acid-etching, SLA) and hydrophilic (SLActive) surfaces to understand the molecular mechanisms responsible for the pro-osteogenic properties of these surfaces. METHODS: The Kyoto Encyclopedia of Genes and Genomes (KEGG® ) pathway database and the Ingenuity® Pathway Analysis (IPA® ) software were applied to the genomic and proteomic data of previous in vivo studies applying SLA and SLActive surfaces, with the specific aim to focus on bone formation-related signalling pathways. While gene data were derived from a human study on osseointegration, protein data originated from a preclinical study in rabbits. Data were available for the 4, 7 and 14 days of healing periods. RESULTS: Both genomic and proteomic data showed that the osteogenesis process takes place mainly at 7 and 14 days of healing on both SLA and SLActive surfaces. Surface hydrophilicity enhances bone formation at multiple levels, by directly promoting an earlier expression of pathways involved in cell proliferation and osteoblast precursor differentiation (eg, mitogen-activated protein kinase, phosphoinositide-3 kinase-AKT, Wnt, Notch, transforming growth factor-ß), but also by positively regulating angiogenesis, bone mineralization and bone remodelling. CONCLUSION: This study combined, for the first time, different 'omics' outputs to get new insights on the molecular mechanisms behind the influence of surface hydrophilicity on osseointegration/bone formation. Specific signalling pathways, such as Wnt, vascular endothelial growth factor and mitogen-activated protein kinase, were identified as differentially modulated by titanium surface hydrophilicity both at a genomic and proteomic level. These findings may be used in the future to monitor/predict the bone formation/osseointegration process, or as a screening tool towards the manufacture of new pro-osteogenic implant surfaces. In order to take into account the full complexity and interplay of cell signalling during bone formation, powerful bioinformatics tools integrating different 'omics' data and predicting signalling pathways trends should be applied by future studies.

Animal models for bisphosphonate-related osteonecrosis of the jaws--an appraisal.

The prolonged use of bisphosphonates has been shown to cause a condition termed 'bisphosphonate related osteonecrosis of the jaws' (BRONJ). BRONJ is a disease entity which has only been described relatively recently, and its multi-factorial aetiology is yet to be fully elucidated. Therefore, the treatment of BRONJ lesions remains a challenge, and animal models are necessary to assist researchers in better understanding the disease. This has led to the recent publication of a number of studies utilising a variety of animal models of BRONJ. This review outlines the factors to be considered when selecting an animal model for BRONJ and discusses the current literature in this rapidly progressing field of research. It is important to consider the applicability of a given model to the clinical condition presenting in humans, and to this end, thorough characterisation of the clinical, histological, radiographic and systemic features is necessary. The development of a clinical lesion is an important consideration in terms of choosing a relevant model, and it appears clear that surgical manipulation, generally involving tooth extraction, is necessary for successful induction of the classic 'clinical' lesion of BRONJ.

Transcriptional profiling of "guided bone regeneration" in a critical-size calvarial defect.

OBJECTIVES: Guided bone regeneration (GBR) is a commonly utilized surgical technique in the craniofacial region. The transcriptional mechanisms associated with this type of bone regeneration are not well understood. The aim of this study was to characterize the transcriptome associated with GBR of a critical-size calvarial defect in the rat. MATERIAL AND METHODS: Critical-size calvarial defects were created in six Wistar strain rats and treated according to the principles of GBR. The tissue filling the regenerating defect was harvested at 7 and 14 days. Total RNA was extracted and microarray analysis was carried out to identify the differences in the transcriptome between days 7 and 14. RESULTS: Gene ontology (GO) analysis of the genes up-regulated at day 7 showed that immature wound healing-related mechanisms, such as protein metabolism and cell proliferation, were up-regulated at this time point. Furthermore, the immuno-inflammatory process was also up-regulated at the earlier time point. In contrast, by day 14, GO groups consistent with wound maturation, such as extracellular matrix formation, anatomical structure development and cell differentiation, were up-regulated. Furthermore, the functionally important GO categories of skeletal development, ossification and bone mineralization were up-regulated at day 14. Genes of interest that belonged to this group and were up-regulated at day 14 included growth and differentiation factors (Bmp2, Bmp3, Tgfb3), extracellular matrix proteins (osteocalcin, osteomodulin, stenniocalcin 1) and transcription factors (Runx2, Sox6, Satb2). Furthermore, a number of genes associated with Tgfß/Bmp and Wnt signalling were also up-regulated. Besides skeletogenesis, genes associated with angiogenesis and neurogenesis were also up-regulated at day 14. CONCLUSIONS: The transcriptome associated with a maturing GBR-treated craniofacial bone defect is characterized by the down-regulation of the immuno-inflammatory response and up-regulation of skeletogeneis-, angiogenesis- and neurogenesis-associated genes. The Tgfß/Bmp and Wnt signalling pathways play an important role in the regenerative process.

In vivo gene expression profile of guided bone regeneration associated with a microrough titanium surface.

OBJECTIVES: To determine the gene expression profile characteristic of "guided bone regeneration" associated with a microrough titanium surface. MATERIAL AND METHODS: Critical-size calvarial defects were treated with the principle of "guided bone regeneration," whereby the extracranial barriers were either polished (SMO) or microrough (SLA) titanium disks. After 7 and 14 days, the contents of the regenerating defect were collected, RNA was extracted and microarray analysis was carried out. At each time point, the healing associated with the microrough surface was compared with that associated with the polished titanium surface. RESULTS: On comparing the SLA and SMO profiles, there were few genes different at day 7 (∼250), whereas there were a large number of genes different at day 14 (∼6500). At day 14, the list of genes that were differentially regulated in response to the SMO and SLA surfaces had an over-representation of genes associated with the functionally relevant gene ontology categories of regeneration, skeletogenesis, mesenchymal cell differentiation, angiogenesis and neurogenesis. There were a greater number of genes within each of these functionally relevant categories that were up-regulated on the SLA surface compared with the SMO surface. The main signalling pathway that was differentially regulated between the two surfaces at day 14 was the Wnt signaling pathway. CONCLUSIONS: Minimal difference was observed between the SMO and the SLA samples at day 7, whereas significant differences were noted at day 14, including genes associated with a number of functionally relevant gene ontology groups. The differentially regulated biological processes provide an insight into the influence of surface topography on "guided bone regeneration" at the cellular and molecular level.

Transcriptional profiling of osseointegration in humans.

OBJECTIVE: To determine the temporal gene expression profile associated with the early healing events during osseointegration in a human model. MATERIAL AND METHODS: Nine solid screw-type cylindrical titanium implants, 4 mm long and 2.8 mm wide, with a chemically modified surface (SLActive) were surgically inserted in the retromolar area of nine human volunteers. The devices were removed using a trephine following 4, 7 and 14 days of healing. The tissue surrounding the implant was harvested, total RNA was extracted and microarray analysis was carried out to identify the differences in the transcriptome between days 4, 7 and 14. RESULTS: Gene ontology (GO) analysis of the temporal transcriptional changes was characteristic of a maturing, osteogenic process over the course of the study (4-14 days). At day 4, a gene expression profile associated with proliferation and immuno-inflammatory processes was predominant. However, by day 14, by far the most predominant mechanisms were associated with skeletogenesis, with the GO categories of skeletal system development, bone development and ossification being predominant, with the majority of changes occurring between days 7 and 14. Furthermore, the biological processes of angiogenesis and neurogenesis were also predominant by day 14. In terms of signal transduction, I-κB kinase/NF-κB cascade was predominant at day 4, whereas TGF-ß/BMP, Wnt and Notch signalling were all associated with the osteogenic process over the duration of the study. Furthermore, Ras and Rho protein signal transduction was regulated throughout the osseointegration process. CONCLUSION: The temporal transcriptional changes during osseointegration involve the expression of proliferation and immuno-inflammatory response associated genes during the early stages of osseointegration, which are ultimately replaced by genes associated with the biological processes of skeletogenesis, angiogenesis and neurogenesis. The early immuno-inflammatory changes appear to be regulated via the I-κB kinase/NF-κB cascade, whereas the later osteogenesis-related mechanisms are regulated by TGF-ß/BMP, Notch and Wnt signaling.

Gene expression profile of osseointegration of a hydrophilic compared with a hydrophobic microrough implant surface.

OBJECTIVES: To compare the gene expression profile of osseointegration associated with a moderately rough and a chemically modified hydrophilic moderately rough surface in a human model. MATERIAL AND METHODS: Eighteen solid screw-type cylindrical titanium implants, 4 mm long and 2.8 mm wide, with either a moderately rough (SLA) or a chemically modified moderately rough (SLActive) surface were surgically inserted in the retromolar area of nine human volunteers. The devices were removed using a trephine following 4, 7 and 14 days of healing. The tissue surrounding the implant was harvested, total RNA was extracted and microarray analysis was carried out to identify the differences in the transcriptome between the SLA and SLActive surfaces at days 4, 7 and 14. RESULTS: There were no functionally relevant gene ontology categories that were over-represented in the list of genes that were differentially expressed at day 4. However, by day 7, osteogenesis- and angiogenesis-associated gene expression were up-regulated on the SLActive surface. Osteogenesis and angiogenesis appeared to be regulated by BMP and VEGF signalling, respectively. By day 14, VEGF signalling remains up-regulated on the SLActive surface, while BMP signalling was up-regulated on the SLA surface in what appeared to be a delayed compensatory response. Furthermore, neurogenesis was a prominent biological process within the list of differentially expressed genes, and it was influenced by both surfaces. CONCLUSIONS: Compared with SLA, SLActive exerts a pro-osteogenic and pro-angiogenic influence on gene expression at day 7 following implant insertion, which may be responsible for the superior osseointegrative properties of this surface.

Progression of periodontal disease and interleukin-10 gene polymorphism.

BACKGROUND AND OBJECTIVE: Interleukin-10 is a key immunoregulatory cytokine that may be of significance in the immunopathogenesis of chronic inflammatory diseases such as periodontal disease. Molecular genetic studies have defined a number of haplotypes that may be associated with differing levels of interleukin-10 secretion. The present study investigated the possible association between interleukin-10 gene polymorphism and periodontal disease progression. MATERIAL AND METHODS: Genomic DNA was obtained from 252 adults who were part of a prospective longitudinal study on the progression of periodontal disease in a general adult Australian population. Single nucleotide polymorphisms at positions -592 and -1082 in the interleukin-10 promoter were analysed using an induced heteroduplex methodology and used to determine interleukin-10 promoter haplotypes in individual samples. Periodontitis progression was assessed by measuring probing depths and relative attachment levels at regular intervals over a 5-year period. A generalized linear model was used to analyse the data, with age, gender, smoking status, interleukin-1 genotype and Porphyromonas gingivalis included as possible confounders. RESULTS: There was a significant (p approximately 0.02) main effect of interleukin-10 haplotypes, with individuals having either the ATA/ACC or the ACC/ACC genotype experiencing around 20% fewer probing depths of >or= 4 mm compared to individuals with other genotypes. Age and smoking had significant (p < 0.001) additional effects. CONCLUSION: These data suggest that the interleukin-10 genotype contributes to the progression of periodontal disease.

A 5-year longitudinal study of Tannerella forsythia prtH genotype: association with loss of attachment.

BACKGROUND: Tannerella forsythia (previously T. forsythensis) in subgingival plaque has been recognized as a defined periodontal pathogen, but its mere presence may be insufficient for disease initiation and/or progression. The organism may produce a cysteine protease, encoded by the prtH gene, which may play a role in the transition from commensal organism to opportunistic pathogen. This study aimed to relate changes in the level of T. forsythia prtH genotype over a 5-year period to a concomitant loss of attachment. METHODS: Real-time polymerase chain reaction was used to quantify the level of the prtH gene in plaque samples from subjects with and without attachment loss (> or =2 mm in at least two sites) over a 5-year period. Clinical measures and subgingival plaque samples were obtained at yearly intervals. RESULTS: Baseline levels of the prtH genotype were significantly lower in the subjects without loss of attachment compared to those who lost attachment over 1, 2, 4, or 5 years. In the subjects with loss of attachment, the higher prtH levels at baseline were not maintained until the end of the observation period. CONCLUSION: Higher levels of the prtH genotype were associated significantly with future attachment loss.

Prostaglandin E2 enhances transforming growth factor-beta 1 and TGF-beta receptors synthesis: an in vivo and in vitro study.

The aims of this study were to determine how Prostaglandin E2 (PGE2) locally applied affected the immunodistribution of latent transforming growth factor-beta 1 (TGF-beta1), and how the eicosanoid modified TGF-beta1 release and TGF-beta receptors gene expression in cultured osteoblasts. PGE2 locally delivered on the rat mandible at doses of 0.1 and 0.05 mg/day, but not 0.025 mg/day, over 20 days significantly increased latent TGF-beta1 immunodistribution (P<0.001), comparing with a placebo-treated group. Cultured osteoblasts stimulated with 10(-5) or 10(-7)M PGE2 significantly varied the level of activated TGF-beta1 released into supernatants at different experimental periods compared with negative and positive controls. TGF-beta receptor type I gene expression was significantly increased in osteoblasts (P<0.01) after 10 days of treatment with 10(-5) and 10(-7)M PGE2, whereas 10(-3) M PGE2 produced the opposite effect. It is concluded that PGE2 may stimulate bone deposition by affecting TGF-beta pathway. This effect on the pathway appears to be dose-dependent.

Larynx motion associated with swallowing during radiation therapy.

PURPOSE: A basis is presented for predicting the reduction in radiation dose to the larynx attributable to swallowing during radiation therapy treatment. METHODS AND MATERIALS: Laryngeal movement associated with swallowing can occur during radiation therapy even when the patient's head is immobilized. Data on the extent and timing of laryngeal motions and the frequency of swallowing were used to predict the effect such motion would have on accuracy of radiation dose to the larynx. RESULTS: In a nontracheostomized adult the larynx elevates approximately 2 cm during a swallow and moves anteriorly less than 1 cm. The normal frequency of swallowing in the supine position is once every 1-2 min. During therapy, the likelihood of a swallow occurring during an irradiation interval depended on the duration of the interval. For irradiation intervals less than 2 min long the ratio of number of swallows to number of intervals was 0.27. For irradiation intervals between 2-3 min long the ratio was 1.76. Based on conservative estimates of radiation field dimensions, larynx motion, and incidence of swallowing, the reduction in radiation dose attributable to swallowing during treatment would be approximately 0.5%. CONCLUSION: With small fields the total dose is only decreased by 0.5% with swallowing, so the change in the total dose is insignificant.

Mastication and swallowing in patients with postirradiation xerostomia.

PURPOSE: Very little objective data has been reported on mastication and swallowing in xerostomic patients, which would substantiate presumed causal relationships between xerostomia and patient complaints. The purpose was to elucidate which components of mastication and swallowing were abnormal, and most directly related to xerostomia, and which appeared unaffected. METHODS AND MATERIALS: A retrospective analysis of timing events in mastication and swallowing was done using videofluoroscopic data for 15 cancer patients with xerostomia, and 20 normal controls. Scintigraphy was also used to determine oropharyngeal residue after a water swallow. Cancer treatment modalities included radiation therapy or chemoradiation therapy. RESULTS: For barium liquid and paste substances, timing measures were equivalent for controls and patients. Xerostomic patients took 46% longer to masticate a shortbread cookie, and timing for the initiation of swallowing was shorter, but duration of swallowing appeared unaffected. Oral and pharyngeal residues following the swallow were greater in the patient group. CONCLUSIONS: Xerostomia primarily affected mastication and oral manipulation of a dry, absorbent food material. Increased oral and pharyngeal residues after a water swallow are ambiguously related to xerostomia. The initiation and duration of the pharyngeal swallow was not abnormal.

Quantifying aspiration in scintigraphic deglutition testing: tissue attenuation effects.

UNLABELLED: Scintigraphic studies for determining aspiration associated with swallowing have ignored error due to differential gamma attenuation in the patient by the various regions of the body. This study sought to estimate the magnitude of that error, and to assess the feasibility of providing individual attenuation corrections based on clinical data. METHODS: Relative attenuation for the pharynx, thorax and abdomen were determined from physical measurements employing an anthropomorphic phantom and 45 adult human subjects. A small sealed radioactive source of 2.5 mCi of 99mTc was placed inside the phantom at various locations within the upper digestive tract and respiratory system, and relative count rates determined via static scans with a gamma camera. Similar data for human subjects was obtained from clinical swallowing testing using a bolus of 2.5 mCi of 99mTc in 10 cc of water. RESULTS: The ratios representing relative counts were highly similar between the phantom and average human data. Test-retest replication of results was good for the abdominal reference and pharynx ratios--less so for the thorax. A procedure is described for estimating accuracy of percent aspiration calculation based on group data, using normalization coefficients derived for separate anatomical regions in the subglottic respiratory system. CONCLUSIONS: Error in percent aspiration calculation will depend on the amount and location of aspirate. Individual subject corrections based on the type of clinical data studied should be attempted with caution.

A microbiological study of Papillon-Lefévre syndrome in two patients.

AIM: To analyse the microflora of subgingival plaque from patients with Papillon-Lefévre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction. METHODS: Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography. Some isolates were also subjected to partial 16S rDNA sequencing. Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies. RESULTS: The culture results showed that most isolates were capnophilic and facultatively anaerobic species-mainly Capnocytophaga spp and Streptococcus spp. The latter included S. constellatus, S. oralis, and S. sanguis. Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus. The aerobic bacteria isolated were species of neisseria and bacillus. Anaerobic species included Prevotella intermedia, P. melaninogenica, and P. nigrescens, as well as Peptostreptococcus spp. ELISA detected P gingivalis in one patient in all sites sampled, whereas A. actinomycetemcomitans was detected in only one site from the other patient. Prevotella intermedia was present in low numbers. CONCLUSIONS: Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens. However, no particular periodontopathogen is invariably associated with PLS.

Persistent colonization with Tannerella forsythensis and loss of attachment in adolescents.

Colonization with Tannerella forsythensis may characterize the conversion of periodontally healthy sites into diseased sites. This three-year study describes the prevalence of T. forsythensis and its relationship to clinical loss of attachment (LOA) in a group of adolescents considered at risk of developing early chronic periodontitis. Adolescents with (LOA+) and without (LOA-) loss of attachment were examined at baseline and 1.5 and 3 yrs subsequently. On each occasion, attachment loss was measured on selected teeth, and the presence of T. forsythensis in their subgingival plaque samples was determined by PCR. T. forsythensis prevalence in LOA+ subjects at baseline (64%) increased to 82% and 86% on subsequent examinations. In contrast, prevalence of T. forsythensis in LOA- subjects was always significantly lower (25%, 36%, and 32%, respectively). The odds of loss of attachment were 8.16 times greater in subjects infected with T. forsythensis at each examination. These results suggest that T. forsythensis is strongly associated with loss of attachment in this adolescent population.

Scintigraphic quantification of aspiration reduction with the Passy-Muir valve.

The Passy-Muir "speaking" tracheostomy valve has been noted to aid in swallowing, based on videofluoroscopy to assess aspiration. In this study scintigraphy was used to quantify the amount of material aspirated. Eleven patients were studied who currently had a tracheostomy in place and were either known to aspirate or were suspected of aspirating. Most were post-treatment head and neck cancer patients who were tumor free at the time of testing. Swallowing was evaluated using videofluoroscopy and scintigraphy. Videofluoroscopy was performed to assess anatomy and determine whether aspiration had occurred. Scintigraphic testing was then performed when the patient had the one-way valve on, and again with it off and tracheostomy open. Following a swallow, the amount (%) of aspirate with the valve in place was found to be significantly less than with the tracheostomy open. A one-way valve can be helpful in reducing aspiration in patients who are at risk for aspiration and who require that their tracheostomy be open.

Scintigraphic assessment of swallow efficiency postlaryngectomy.

There have been reports of a high incidence of hypopharyngeal stenosis in total laryngectomy patients when the surgery requires a partial pharyngectomy for pyriform sinus involvement. In this study, three groups were compared: total laryngectomy patients without partial pharyngectomy, total laryngectomy patients with partial pharyngectomy, and normal controls. All patients had received radiation therapy following surgery. All were maintaining oral nutrition, and none complained of dysphagia. Patients were tested between 1 and 7 months postradiation therapy, with a mean of 3 months. Measures of swallowing efficiency were based on scintigraphic data for a liquid swallow. Patients with partial pharyngectomy had abnormally long oropharyngeal transit times and low efficiency scores. For a subgroup of patients with partial pharyngectomy, swallowing data were available postsurgery and postradiation therapy. Postsurgery this patient group did not differ significantly from normal patients in swallowing efficiency, and swallowing efficiency deteriorated in postradiation therapy. This scintigraphic methodology is shown to be a sensitive method of assessing swallowing function in this patient population.

The effect of swallowing bolus and head position on eustachian tube function via sonotubometry.

Eustachian tube function was evaluated by sonotubometry in otologically normal young adults. The purpose of the research was to determine if the incidence or duration of eustachian tube opening would be altered with changes in swallowing bolus volume or changes in head rotation. The subject performed multiple swallows of 10 cc water bolus and dry swallows (saliva), with the head in the straight-forward, turned right, and turned left positions. Both ears were evaluated. The average incidence and duration of eustachian tube opening for all swallows was 74% (398 msec). No significant differences were found with any combination of bolus type or head position, suggesting that neither liquid bolus volume nor head rotation need be controlled when sonotubometry is performed.

Interpreting the sounds of swallowing: fluid flow through the cricopharyngeus.

The sounds of swallowing in normal subjects were recorded by means of a miniature accelerometer on the throat, simultaneously with videofluoroscopy of a modified barium swallow. Consistent correspondence was found between a rapid change in the acoustic spectrum of the accelerometer signal (the appearance of a region of energy prominence above 1,000 Hz) and bolus flow through the cricopharyngeus.

Bovine ocular squamous cell carcinoma: superoxide dismutase and catalase levels.

Considerable evidence exists for the production of free radicals in the eye region after exposure to ultraviolet light. Failure to remove these radicals adequately may be important in the predisposition of Hereford cattle to ocular carcinoma. The levels of two enzymes, superoxide dismutase and catalase, involved in the removal of superoxide anion and hydrogen peroxide, were compared in Hereford cattle predisposed to ocular carcinoma and a resistant breed, Droughtmaster cattle. The results demonstrate comparable levels of superoxide dismutase in the two breeds in both eye tissue and erythrocytes. Catalase levels also were the same in both breeds. Increased incidence of ocular carcinoma in Herefords does not appear to be due to a deficiency in either superoxide dismutase or catalase.