Reconstructive Microsurgery and Tissue Engineering in Musculo-Skeletal Oncology - Innovative Techniques

Tumors involving bone and soft tissues are extremely challenging situations. With the recent advances of multi-modal treatment, not only the type of surgery has moved from amputation to limb-sparing procedures, but also the survivorship has improved considerably and reconstructive techniques have the goal to allow a considerably higher quality of life. In bone reconstruction, tissue engineering strategies are the main area of research. Re-vascularization and re-vitalisation of a massive allograft would considerably improve the outcome of biological reconstructions. Using a rabbit animal model, in this study we showed that, by implanting a vascular pedicle inside a weight bearing massive cortical allograft, the bone regeneration inside the allograft was higher compared to the non-vascularized implants, given the patency of the vascular pedicle. Improvement in the animal model and the addition of Stem Cells and Growth factors will allow a further improvement in the results. In soft tissue tumors, free and pedicled flaps have been proven to be of great help as reconstruction strategies. In this study we analyzed the functional and overall outcome of 14 patients who received a re-innervated vascularized flap. We have demonstrated that the use of the innovative technique of motor re-innervated muscular flaps is effective when the resection involves important functional compartments of the upper or lower limb, with no increase of post-operative complications. Although there was no direct comparison between this type of reconstruction and the standard non-innervated reconstruction, we underlined the remarkable high overall functional scores and patient satisfaction following this procedure.

Correlation of fat embolism severity and subcutaneous fatty tissue crushing and bone fractures

Pulmonary fat embolism (PFE) is frequently encountered in blunt trauma. The clinical manifestation ranges from no impairment in light cases to death due to right-sided heart failure or hypoxaemia in severe cases. Occasionally, pulmonary fat embolism can give rise to a fat embolism syndrome (FES), which is marked by multiorgan failure, respiratory disorders, petechiae and often death. It is well known that fractures of long bones can lead to PFE. Several authors have argued that PFE can arise due to mere soft tissue injury in the absence of fractures, a claim other authors disagree upon. In this study, we retrospectively examined 50 victims of blunt trauma with regard to grade and extent of fractures and crushing of subcutaneous fatty tissue and presence and severity of PFE. Our results indicate that PFE can arise due to mere crushing of subcutaneous fat and that the fracture grade correlated well with PFE severity (p = 0.011). The correlation between PFE and the fracture severity (body regions affected by fractures and fracture grade) showed a lesser significant correlation (p = 0.170). The survival time (p = 0.567), the amount of body regions affected by fat crushing (p = 0.336) and the fat crush grade (p = 0.485) did not correlate with the PFE grade, nor did the amount of body regions affected by fractures. These results may have clinical implications for the assessment of a possible FES development, as, if the risk of a PFE is known, preventive steps can be taken.

Patient-centered outcomes after subepithelial connective tissue grafts and coronally advanced flaps

The use of a coronally advanced flap (CAF) and connective tissue graft (CTG) is a well-established procedure to cover single and multiple recessions and improve soft tissue esthetics. However, until now, there are still limited data evaluating patient morbidity, the fear of imminent tooth loss, and modification of sensitivity in surgical areas. The aim of the present study was to evaluate the patient-centered outcomes associated with CAF + CTG.

Reduction of postmortem angiography-induced tissue edema by using polyethylene glycol as a contrast agent dissolver

Postmortem investigation is increasingly supported by computed tomography (CT) and magnetic resonance imaging, in which postmortem minimal invasive angiography has become important. The newly introduced approach using an aqueous contrast agent solution provided excellent vessel visualization but was suspected to possibly cause tissue edema artifacts in histological investigations. The aim of this study was to investigate on a porcine heart model whether it is possible to influence the contrast agent distribution within the soft tissue by changing its viscosity by dissolving the contrast agent in polyethylene glycol (PEG) as a matrix medium. High-resolution CT scans after injection showed that viscosities above c. 15 mPa s (65% PEG) prevented a contrast agent distribution within the capillary bed of the left ventricular myocardium. Thereby, the precondition of edema artifacts could be reduced. Its minimal invasive application on human corpses needs to be further adapted as the flow resistance is expected to differ between different tissues.

Potential of chemically modified hydrophilic surface characteristics to support tissue integration of titanium dental implants

In the past, several modifications of specific surface properties such as topography, structure, chemistry, surface charge, and wettability have been investigated to predictably improve the osseointegration of titanium implants. The aim of the present review was to evaluate, based on the currently available evidence, the impact of hydrophilic surface modifications of titanium for dental implants. A surface treatment was performed to produce hydroxylated/hydrated titanium surfaces with identical microstructure to either acid-etched, or sand-blasted, large grit and acid-etched substrates, but with hydrophilic character. Preliminary in vitro studies have indicated that the specific properties noted for hydrophilic titanium surfaces have a significant influence on cell differentiation and growth factor production. Animal experiments have pointed out that hydrophilic surfaces improve early stages of soft tissue and hard tissue integration of either nonsubmerged or submerged titanium implants. This data was also corroborated by the results from preliminary clinical studies. In conclusion, the present review has pointed to a potential of hydrophilic surface modifications to support tissue integration of titanium dental implants.

Stability of hard tissue profile after mandibular setback in sagittal split osteotomies: a longitudinal and long-term follow-up study

The aim of the study was to conduct a long-term follow-up on the stability of the hard tissues after bilateral sagittal split osteotomy (BSSO) with rigid internal fixation (RIF)to set back the mandible and to compare it with that of mandibular advancement performed by the same team of surgeons and with the same examination protocol. Seventeen consecutive patients (6 females and 11 males) could be re-examined 12.7 years (T5) after surgery. The previous examinations were before surgery (T1), 5 days (T2), and 6.6 (T3) and 14.4 (T4) months after surgery. Lateral cephalograms were traced by hand, digitized, and evaluated with the Dentofacial Planner software program. The x-axis for the system of co-ordinates ran through sella (point zero) and the line nasion-sella-line minus 7 degrees. The program determined the x- and y-values of each variable and the usual angles and distances. The effects of treatment were determined with Wilcoxon matched pairs, signed ranks test, with Bonferroni adjustment, and the relationship between variables with Spearman rank correlation coefficient. Relapse at point B was 0.94 mm or 15 per cent and at pogonion 1.46 mm or 21 per cent of the initial setback at T5. Relapse was mainly short-term (T4-T2), 13 per cent for point B and 17 per cent for pogonion. Gender correlated significantly with relapse (T5-T2) at point B (P = 0.002) and pogonion (P = 0.021), i.e. females in contrast to males showed further distalization of the mandible instead of relapse. No correlations were seen for age or the amount of surgical setback. The long-term results in mandibular setback patients were more stable when compared with the mandibular advancement patients examined previously. The initial soft tissue profile, the initial growth direction, and the remodelling processes of the hard tissues must be considered as reasons for long-term relapse. Growth direction positively influenced the long-term results in females: further distalization of the mandible occurred.

Three-dimensional imaging methods for quantitative analysis of facial soft tissues and skeletal morphology in patients with orofacial clefts: a systematic review.

BACKGROUND Current guidelines for evaluating cleft palate treatments are mostly based on two-dimensional (2D) evaluation, but three-dimensional (3D) imaging methods to assess treatment outcome are steadily rising. OBJECTIVE To identify 3D imaging methods for quantitative assessment of soft tissue and skeletal morphology in patients with cleft lip and palate. DATA SOURCES Literature was searched using PubMed (1948-2012), EMBASE (1980-2012), Scopus (2004-2012), Web of Science (1945-2012), and the Cochrane Library. The last search was performed September 30, 2012. Reference lists were hand searched for potentially eligible studies. There was no language restriction. STUDY SELECTION We included publications using 3D imaging techniques to assess facial soft tissue or skeletal morphology in patients older than 5 years with a cleft lip with/or without cleft palate. We reviewed studies involving the facial region when at least 10 subjects in the sample size had at least one cleft type. Only primary publications were included. DATA EXTRACTION Independent extraction of data and quality assessments were performed by two observers. RESULTS Five hundred full text publications were retrieved, 144 met the inclusion criteria, with 63 high quality studies. There were differences in study designs, topics studied, patient characteristics, and success measurements; therefore, only a systematic review could be conducted. Main 3D-techniques that are used in cleft lip and palate patients are CT, CBCT, MRI, stereophotogrammetry, and laser surface scanning. These techniques are mainly used for soft tissue analysis, evaluation of bone grafting, and changes in the craniofacial skeleton. Digital dental casts are used to evaluate treatment and changes over time. CONCLUSION Available evidence implies that 3D imaging methods can be used for documentation of CLP patients. No data are available yet showing that 3D methods are more informative than conventional 2D methods. Further research is warranted to elucidate it.

Healing of localized gingival recessions treated with coronally advanced flap alone or combined with either a resorbable collagen matrix or subepithelial connective tissue graft: A preclinical study.

OBJECTIVES To histologically evaluate the effectiveness of a porcine derived collagen matrix (CM) and a subepithelial connective tissue graft (CTG) for coverage of localized gingival recessions. MATERIALS AND METHODS Chronic single Miller Class I-like recessions were created at the buccal at the canines and at the third and fourth premolars in the upper and lower jaws of six beagle dogs. The defects were randomly treated with (1) coronally advanced flap surgery (CAF) + CM, (2) CAF + CTG, or (3) CAF alone. At 12 weeks, histometric measurements were made, e.g., between a reference point (N) - and the gingival margin (GM) - and the outer contour of the adjacent soft tissue (gingival thickness [GT]). RESULTS The postoperative healing was uneventful in all animals. No complications such as allergic reactions, abscesses or infections were noted throughout the entire study period. All three treatments resulted in coverage of localized gingival recessions. The histological analysis failed to identify any residues of CM or CTG. The histometric measurements revealed comparable outcomes for N-GM and GT values for all three groups (CAF + CM: 1.04 ± 0.69 mm/0.68 ± 0.33 mm; CAF + CTG: 1.15 ± 1.12 mm/0.76 ± 0.37 mm; CAF: 1.43 ± 0.45 mm/0.79 ± 0.24 mm). CONCLUSIONS In the used defect model, the application of CTG or CM in conjunction with CAF did not have an advantage over the use of CAF alone. CLINICAL RELEVANCE The use of CAF alone is a valuable option for the treatment localized Miller Class I recessions.

A prospective, randomized, controlled study using OsseoSpeed™ implants placed in maxillary fresh extraction socket: soft tissues response

PURPOSE The aim of this work was to study the peri-implant soft tissues response, by evaluating both the recession and the papilla indexes, of patients treated with implants with two different configurations. In addition, data were stratified by tooth category, smoking habit and thickness of buccal bone wall. MATERIALS AND METHODS The clinical trial was designed as a prospective, randomized-controlled multicenter study. Adults in need of one or more implants replacing teeth to be removed in the maxilla within the region 15-25 were recruited. Following tooth extraction, the site was randomly allocated to receive either a cylindrical or conical/cylindrical implant. The following parameters were studied: (i) Soft tissue recession (REC) measured by comparing the gingival zenith (GZ) score at baseline (permanent restoration) with that of the yearly follow-up visits over a period of 3 years (V1, V2 and V3). (ii) Interdental Papilla Index (PI): PI measurements were performed at baseline and compared with that of the follow-up visits. In addition, data were stratified by different variables: tooth category: anterior (incisors and canine) and posterior (first and second premolar); smoking habit: patient smoker (habitual or occasional smoker at inclusion) or non-smoker (non-smoker or ex-smoker at inclusion) and thickness of buccal bone wall (TB): TB ≤ 1 mm (thin buccal wall) or TB > 1 mm (thick buccal wall). RESULTS A total of 93 patients were treated with 93 implants. At the surgical re-entry one implant was mobile and then removed; moreover, one patient was lost to follow-up. Ninety-one patients were restored with 91 implant-supported permanent single crowns. After the 3-year follow-up, a mean gain of 0.23 mm of GZ was measured; moreover, 79% and 72% of mesial and distal papillae were classified as >50%/ complete, respectively. From the stratification analysis, not significant differences were found between the mean GZ scores of implants with TB ≤ 1 mm (thin buccal wall) and TB > 1 mm (thick buccal wall), respectively (P < 0.05, Mann-Whitney U-test) at baseline, at V1, V2 and V3 follow-up visits. Also, the other variables did not seem to influence GZ changes over the follow-up period. Moreover, a re-growth of the interproximal mesial and distal papillae was the general trend observed independently from the variables studied. CONCLUSIONS Immediate single implant treatment may be considered a predictable option regarding soft tissue stability over a period of 3 years of follow-up. An overall buccal soft tissue stability was observed during the GZ changes from the baseline to the 3 years of follow-up with a mean GZ reduction of 0.23 mm. A nearly full papillary re-growth can be detectable over a minimum period of 2 years of follow-up for both cylindrical and conical/cylindrical implants. Both the interproximal papilla filling and the midfacial mucosa stability were not influenced by variables such as type of fixture configuration, tooth category, smoke habit, and thickness of buccal bone wall of ≤ 1 mm (thin buccal wall).

New osseous soft markers for trisomy 13, 18 and 21

INTRODUCTION For ultrasonographic diagnosis of a fetal trisomy so-called "soft markers" (=ultrasonographically detectable morphological variants) are used. Detection of a certain number of them increases the diagnostic certainty of a fetal trisomy. Up to now there are very few diagnostically accepted osseous soft markers for trisomy. Hence potential osseous soft markers applicable for first and second trimester ultrasound screening for trisomy 21, 18 or 13 were studied. METHODS Postmortal fetal X-rays (ap, lateral) of 358 fetuses (trisomy 21: n = 109, trisomy 18: n = 46; trisomy 13: n = 38, control group: n = 165). RESULTS Not yet described but with trisomy 21 statistically associated soft markers were un-timely os sternale ossification, delayed os sacrum ossification, shortened os maxillare, reduced os maxillare-jaw-corner distance, augmented orbita height, premature os calcaneus ossification, bell-shaped thorax, coronal clefts, trend to wider binocular as well as wider intraocular distances; for trisomy 18: elevated clavicula slope, reduced number of ribs, bell-shaped thorax, coronal clefts, reduced os maxillare-jaw-corner distance, shortened ramus mandibulare, shortened os metacarpale IV and V, augmented ratio between biparietal diameter and (osseus and soft-tissue) shoulder width; for trisomy 13: longer os nasale, elevated clavicula slope, premature sternum, delayed os sacrum ossification, delayed/premature cranium ossification, reduced number of ribs, coronal clefts, reduced os maxillare-jaw-corner distance, shortened ramus mandibulare, augmented orbita height, shortened os metacarpale V and a tendency for a shortened os metacarpale IV. CONCLUSION We found several not yet published osseous soft markers statistically associated with trisomy 21, 18 and 13, which can help to ensure sonographically these aneuploidy diagnoses.

A mechanical characterization of soft tissues based on their mesostructure

Se presenta una relación entre las propiedades mecánicas de los tejidos fibrados y las características geométricas y mecánicas de los fibriles que lo forman a escala mesoscópica. In this work we derive a relationship between the mechanical and geometrical properties of the fibril constituents and the soft tissue material parameters at macroscopic scale.

Calcium isotope fractionation between soft and mineralized tissues as a monitor of calcium use in vertebrates

Calcium from bone and shell is isotopically lighter than calcium of soft tissue from the same organism and isotopically lighter than source (dietary) calcium. When measured as the 44Ca/40Ca isotopic ratio, the total range of variation observed is 5.5‰, and as much as 4‰ variation is found in a single organism. The observed intraorganismal calcium isotopic variations and the isotopic differences between tissues and diet indicate that isotopic fractionation occurs mainly as a result of mineralization. Soft tissue calcium becomes heavier or lighter than source calcium during periods when there is net gain or loss of mineral mass, respectively. These results suggest that variations of natural calcium isotope ratios in tissues may be useful for assessing the calcium and mineral balance of organisms without introducing isotopic tracers.

Deficiency of iNOS contributes to Porphyromonas gingivalis-induced tissue damage

Periodontitis is a chronic inflammatory disease that results in extensive soft and hard tissue destruction of the periodontium. Porphyromonas gingivalis possesses an array of virulence factors and has been shown to induce expression of inducible nitric oxide synthase (iNOS) in inflammatory cells. The aim of this study was to investigate the effect of eliminating iNOS in a murine model of P. gingivalis infection. This was achieved by utilizing a P. gingivalis-induced skin abscess model, and an alveolar bone loss model employing an oral infection of P. gingivalis in iNOS knockout mice. The results indicated that iNOS knockout mice exhibit more extensive soft tissue damage and alveolar bone loss in response to P. gingivalis infection compared to wild-type mice. The local immune response to P. gingivalis in iNOS knockout mice was characterized by increased numbers of polymorphonuclear monocytes, while the systemic immune response was characterized by high levels of interleukin-12. The iNOS is required for an appropriate response to P. gingivalis infection.

Novel biodegradable fibres for applications in tissue engineering and drug delivery

The preparation and characterisation of novel biodegradable polymer fibres for application in tissue engineering and drug delivery are reported. Poly(e-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. The tensile strength and stiffness of as-spun fibres were highly dependent on the concentration of the spinning solution. Use of a 6% w/v solution resulted in fibres having strength and stiffness of 1.8 MPa and 0.01 GPa respectively, whereas these values increased to 9.9 MPa and 0.1 GPa when fibres were produced from 20% w/v solutions. Cold drawing to an extension of 500% resulted in further increases in fibre strength (up to 50 MPa) and stiffness (0.3 GPa). Hot drawing to 500% further increased the fibre strength (up to 81 MPa) and stiffness (0.5 GPa). The surface morphology of as-spun fibres was modified, to yield a directional grooved pattern by drying in contact with a mandrel having a machined topography characterised by a peak-peak separation of 91 mm and a peak height of 30 mm. Differential scanning calorimetery (DSC) analysis of as-spun fibres revealed the characteristic melting point of PCL at around 58°C and a % crystallinity of approximately 60%. The biocompatibility of as-spun fibres was assessed using cell culture. The number of attached 3T3 Swiss mouse fibroblasts, C2C12 mouse myoblasts and human umbilical vein endothelial cells (HUVECs) on as-spun, 500% cold drawn, and gelatin coated PCL fibres were observed. The results showed that the fibres promoted cell proliferation for 9 days in cell culture and was slightly lower than on tissue culture plastic. The morphology of all cell lines was assessed on the various PCL fibres using scanning electron microscopy. The cell function of HUVECs growing on the as-spun PCL fibres was evaluated. The ability HUVECs to induce an immune response when stimulated with lipopolysaccaride (LPS) and thereby to increase the amount of cell surface receptors was assessed by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that PCL fibres did not inhibit this function compared to TCP. As-spun PCL fibres were loaded with 1 % ovine albumin (OVA) powder, 1% OVA nanoparticles and 5% OVA nanoparticles by weight and the protein release was assessed in vitro. PCL fibres loaded with 1 % OVA powder released 70%, 1% OVA nanoparticle released 60% and the 5% OVA nanoparticle released 25% of their protein content over 28 days. These release figures did not alter when the fibres were subjected to lipase enzymatic degradation. The OVA released was examined for structural integrity by SDS-PAGE. This showed that the protein molecular weight was not altered after incorporation into the fibres. The bioactivity of progesterone was assessed following incorporation into PCL fibres. Results showed that the progesterone released had a pronounced effect on MCF-7 breast epithelial cells, inhibiting their proliferation. The PCL fibres display high fibre compliance, a potential for controlling the fibre surface architecture to promote contact guidance effects, favorable proliferation rate of fibroblasts, myoblasts and HUVECs and the ability to release pharmaceuticals. These properties recommended their use for 3-D scaffold production in soft tissue engineering and the fibres could also be exploited for controlled presentation and release of biopharmaceuticals such as growth factors.

Tissue-specific bioscaffolds for adipose-derived stem/stromal cell expansion and delivery

Decellularized adipose tissue (DAT) is a promising biomaterial for soft tissue regeneration, and it provides a highly conducive microenvironment for human adipose-derived stem/stromal cell (ASC) attachment, proliferation, and adipogenesis. This thesis focused on developing techniques to fabricate 3-D bioscaffolds from enzymatically-digested DAT as platforms for ASC culture and delivery in adipose tissue engineering and large-scale ASC expansion. Initial work investigated chemically crosslinked microcarriers fabricated from pepsin-digested DAT as injectable adipo-inductive substrates for ASCs. DAT microcarriers highly supported ASC adipogenesis compared to gelatin microcarriers in a CELLSPIN system, as confirmed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, lipid accumulation, and endpoint RT-PCR. ASCs cultured on DAT microcarriers in proliferation medium also had elevated PPARγ, C/EBPα, and LPL expression which suggested adipo-inductive properties. In vivo testing of the DAT microcarriers exhibited stable volume retention and enhanced cellular infiltration, tissue remodeling, and angiogenesis. Building from this work, non-chemically crosslinked porous foams and bead foams were fabricated from α-amylase-digested DAT for soft tissue regeneration. Foams were stable and strongly supported ASC adipogenesis based on GPDH activity and endpoint RT-PCR. PPARγ, C/EBPα, and LPL expression in ASCs cultured on the foams in proliferation media indicated adipo-inductive properties. Foams with Young’s moduli similar to human fat also influenced ASC adipogenesis by enhanced GPDH activity. In vivo adipogenesis accompanied by a potent angiogenic response and rapid resorption showed their potential use in wound healing applications. Finally, non-chemically crosslinked porous microcarriers synthesized from α-amylase-digested DAT were investigated for ASC expansion. DAT microcarriers remained stable in culture and supported significantly higher ASC proliferation compared to Cultispher-S microcarriers in a CELLSPIN system. ASC immunophenotype was preserved for all expanded groups, with reduced adhesion marker expression under dynamic conditions. DAT microcarrier expansion upregulated ASC expression of early adipogenic (PPARγ, LPL) and chondrogenic (COMP) markers without inducing a mature phenotype. DAT microcarrier expanded ASCs also showed similar levels of adipogenesis and osteogenesis compared to Cultispher-S despite a significantly higher population fold-change, and had the highest level of chondrogenesis among all groups. This study demonstrates the promising use of DAT microcarriers as a clinically relevant strategy for ASC expansion while maintaining multilineage differentiation capacity.