Is remaining intervertebral disc tissue interfering with bone generation during fusion of two vertebrae?

STUDY DESIGN: laboratory research. BACKGROUND: Through the increasing number of minimally invasive procedures in spinal fusion surgery, the complete removal of intervertebral disc (IVD) tissue has become more a challenge. Remaining IVD may interfere with the biological process of bone formation. OBJECTIVE: In order to establish whether complete removal of IVD tissue will improve or inhibit the fusion process, the effects of different concentrations of extracts of inflamed disc tissue on the mitochondrial activity of mesenchymal stem cells (MSCs), and the capacity to mineralize their extracellular matrix by osteoblasts and differentiated MSCs were tested in vitro. METHODS: A MTT assay was conducted to measure the mitochondrial activity of MSCs, and an Alizarin Red S staining quantification assay to measure the deposition of calcium by osteoblasts and differentiated, bone marrow-derived MSCs. RESULTS: A significantly higher mitochondrial activity was shown in MSCs co-cultured with extracts of IVD tissue (10%, 50%, and 100%) compared with the control group after 48 hours of incubation, indicating that the IVD tissue extracts stimulated the mitochondrial activity of MSCs. This effect appeared to be inversely proportional to the concentration of IVD tissue extract. No significant differences in mineralization by human osteoblasts or differentiated MSCs were found between the samples incubated with IVD tissue extracts (3% and 33%) and the control samples. CONCLUSION: Our findings indicate that remaining IVD tissue has more of a stimulating than inhibiting effect on the activity of MSCs. Even if inflammatory cytokines are produced, these do not result in a net inhibition of cellular activity or osteogenic differentiation of MSCs.

The implant infection paradox: why do some succeed when others fail? Opinion and discussion paper.

Biomaterial-implants are frequently used to restore function and form of human anatomy. However, the presence of implanted biomaterials dramatically elevates infection risk. Paradoxically, dental-implants placed in a bacteria-laden milieu experience moderate failure-rates, due to infection (0.0-1.1%), similar to the ones of joint-arthroplasties placed in a near-sterile environment (0.1-1.3%). Transcutaneous bone-fixation pins breach the immune-barrier of the epidermis, exposing underlying sterile-tissue to an unsterile external environment. In contrast to dental-implants, also placed in a highly unsterile environment, these pins give rise to relatively high infection-associated failure-rates of up to 23.0%. Herein, we attempt to identify causes as to why dental-implants so often succeed, where others fail. The major part of all implants considered are metal-made, with similar surface-finishes. Material choice was therefore discarded as underlying the paradox. Antimicrobial activity of saliva has also been suggested as a cause for the success of dental-implants, but was discarded because saliva is the implant-site-fluid from which viable bacteria adhere. Crevicular fluid was discarded as it is largely analogous to serum. Instead, we attribute the relative success of dental-implants to (1) ability of oral tissues to heal rapidly in the continuous presence of commensal bacteria and opportunistic pathogens, and (2) tolerance of the oral immune-system. Inability of local tissue to adhere, spread and grow in presence of bacteria and an intolerant immune-system are identified as the likely main causes explaining the susceptibility of other implants to infection-associated failure. In conclusion, it is the authors' belief that new anti-infection strategies for a wide range of biomaterial-implants may be derived from the relative success of dental-implants.

Tissue-engineered constructs: the effect of scaffold architecture in osteochondral repair.

Cartilage has a poor regenerative capacity. Tissue-engineering approaches using porous scaffolds seeded with chondrocytes may improve cartilage repair. The aim of this study was to examine the effect of pore size and pore interconnectivity on cartilage repair in osteochondral defects treated with different scaffolds seeded with allogenic chondrocytes. Scaffolds consisting of 55 wt% poly(ethylene oxide terephthalate) and 45 wt% poly(butylene terephthalate) (PEOT/PBT) with different pore sizes and interconnectivities were made, using a compression moulding (CM) and a three-dimensional fibre (3DF) deposition technique. In these scaffolds, allogenic chondrocytes were seeded, cultured for 3 weeks and implanted in osteochondral defects of skeletally mature rabbits. At 3 weeks no difference in cartilage repair between an empty osteochondral defect, CM or 3DF scaffolds was found. Three months post-implantation, cartilage repair was significantly improved after implantation of a 3DF scaffold compared to a CM scaffold. Although not significant, Mankin scores for osteoarthritis (OA) indicated less OA in the 3DF scaffold group compared to empty defects and CM-treated defects. It is concluded that scaffold pore size and pore interconnectivity influences osteochondral repair and a decreased pore interconnectivity seems to impair osteochondral repair.

Bacterial biofilm formation versus mammalian cell growth on titanium-based mono- and bi-functional coating.

Biomaterials-associated-infections (BAI) are serious complications in modern medicine. Although non-adhesive coatings, like polymer-brush coatings, have been shown to prevent bacterial adhesion, they do not support cell growth. Bi-functional coatings are supposed to prevent biofilm formation while supporting tissue integration. Here, bacterial and cellular responses to poly(ethylene glycol) (PEG) brush-coatings on titanium oxide presenting the integrin-active peptide RGD (arginine-glycine-aspartic acid) (bioactive "PEG-RGD") were compared to mono-functional PEG brush-coatings (biopassive "PEG") and bare titanium oxide (TiO2) surfaces under flow. Staphylococcus epidermidis ATCC 35983 was deposited on the surfaces under a shear rate of 11 s-1 for 2 h followed by seeding of U2OS osteoblasts. Subsequently, both S. epidermidis and U2OS cells were grown simultaneously on the surfaces for 48 h under low shear (0.14 s-1). After 2 h, staphylococcal adhesion was reduced to 3.6-/+1.8 x 103 and 6.0-/+3.9 x 103 cm-2 on PEG and PEG-RGD coatings respectively, compared to 1.3-/+0.4 x 105 cm-2 for the TiO2 surface. When allowed to grow for 48 h, biofilms formed on all surfaces. However, biofilms detached from the PEG and PEG-RGD coatings when exposed to an elevated shear (5.6 s-1) U2OS cells neither adhered nor spread on PEG brush-coatings, regardless of the presence of biofilm. In contrast, in the presence of biofilm, U2OS cells adhered and spread on PEG-RGD coatings with a significantly higher surface coverage than on bare TiO2. The detachment of biofilm and the high cell surface coverage revealed the potential significance of PEG-RGD coatings in the context of the "race for the surface" between bacteria and mammalian cells.

The effects of different decalcification protocols on TUNEL and general cartilage staining.

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.

Anxiety and self-management behaviour in chronic obstructive pulmonary disease: what has been learned?

A considerable amount of literature has described the prevalence of anxiety in patients with the lung condition chronic obstructive pulmonary disease (COPD). Few, if any, papers have reviewed the interrelationship between anxiety symptoms and self-management interventions in this population. This is the aim of the current review. First, the review examines the evidence suggesting that anxiety is more common in COPD than other populations. Secondly, the focus shifts to evaluating the evidence for and against the efficacy of COPD self-management programmes. Finally this paper examines the relationship between anxiety and COPD self-management with particular reference to the benefits and possible harm of some COPD self-management goals and anxious patients.

Couples dealing with cancer: role and gender differences regarding psychological distress and quality of life.

The goal of the present study was to further knowledge on gender and role (i.e. patient versus partner) differences in psychological distress and quality of life as a consequence of dealing with cancer. There is some evidence that being the patient or the caregiver makes more difference for men than for women. In total, 173 couples facing various forms of cancer (two samples) and a control group of 80 couples completed the CES-D and Cantril's Ladder. Analyses of variance revealed that both female patients and female partners of patients perceived more psychological distress and a lower quality of life than women in healthy couples. In contrast, role did have an effect on men. Specifically, male patients scored as high on psychological distress and as low on quality of life as female patients and female partners, but psychological distress and quality of life did not differ between male partners of patients and their healthy controls. However, this effect was found in only one patient sample. The finding that female partners perceived more psychological distress and a lower quality of life than male partners could not be accounted for by differences in the physical condition of the patient or the partner.

Marital satisfaction in patients with cancer: does support from intimate partners benefit those who need it the most?

This cross-sectional study assessed 3 ways of providing spousal support. Active engagement means involving the patient in discussions and using constructive problem-solving methods; protective buffering means hiding one's concerns; and overprotection refers to underestimation of the patient's capabilities, resulting in unnecessary help and excessive praise for accomplishments. Ratings of received spousal support by 68 patients with cancer revealed findings similar to those of partners' ratings of provided support. The positive association between active engagement and the patient's marital satisfaction was stronger for patients with a rather poor psychological and physical condition than for those with a rather good condition. Furthermore, protective buffering and overprotection were negatively associated with marital satisfaction only when patients experienced relatively high levels of psychological distress or physical limitations.

Biodegradable three-dimensional networks of poly(dimethylamino ethyl methacrylate). Synthesis, characterization and in vitro studies of structural degradation and cytotoxicity.

In ophthalmology, there is a need for novel degradable biomaterials for e.g. controlled drug release in the vitreous body. These degradable materials should feature both excellent biocompatibility, and well-defined kinetics of degradation. In most cases, poly(D,L-lactic acid), or poly(lactic-co-glycolic acid) are used. These materials, however, suffer from some serious drawbacks, since the degradation kinetics are difficult to control, especially since the so-called 'burst-degradation' occurs. Here, we describe a set of novel polymeric networks which largely consist of poly(dimethylamino ethyl methacrylate) (poly(DMAEMA)); these materials are crosslinked via a dimethacrylate molecule that contains two carbonate groups. This system is susceptible to hydrolytic scission. The degradation products do not exert a catalytic effect on the ongoing degradation reaction (i.e. there is no burst effect). We describe the synthesis of three of these materials, which differ merely with regard to the crosslinker content. These materials were characterized through DMTA, 1H NMR and FT-IR spectroscopy, and scanning electron microscopy. The reaction DMAEMA + 2-hydroxyethyl methacrylate (HEMA) was studied in detail, using 1H NMR spectroscopy, and these experiments revealed that the reaction of DMAEMA and HEMA produces a random (Bernouillian-type) copolymer. From this, we contend that the new materials have more or less uniform distribution of the crosslinks throughout their volume. Structural degradation of the three materials was studied in vitro, at pH 7.4, 9.1 and 12.0. It is found that the materials exhibit smooth hydrolysis, which can be controlled via the crosslink density and the pH, as was expected a priori. It should be noted that degradation of these materials produces non-hydrolysable, but water-soluble, oligo(DMAEMA) and poly(DMAEMA) molecules. We subsequently performed in vitro studies on the biocompatibility of these materials. The MTT cytotoxicity assay revealed that the materials were cytotoxic to chondrosarcoma cells. This is most probably due to local increase of the pH due to the basic character of the pending dimethylamino groups. Cytotoxicity remained virtually unchanged after extended washing with water. This indicates that the cytotoxicity is an intrinsic property of the material and was not caused by remnants of free monomer. Cytotoxicity was also seen in cell cultures (human fibroblasts isolated from donor corneas) which were grown in contact with the materials. It is concluded that the new materials have attractive degradation characteristics, but their cytotoxicity makes them unsuitable for applications in ophthalmology.

Tailoring of new polymeric biomaterials for the repair of medium-sized corneal perforations.

The aim of this study was to investigate whether polymeric biomaterials can be designed such that they become suitable for surgical closure of medium-sized perforations in the cornea, the transparent tissue in the front of the eye. Such a biomaterial must meet stringent requirements in terms of hydrophilicity, strength, transparency, flexibility, and biocompatibility. Four different copolymers of n-butyl methacrylate (BMA) and hexa(ethylene glycol) methacrylate (HEGMA) were prepared and characterized. Poly(BMA) was made as a reference material. Physicochemical properties were measured (contact angles, glass-transition temperatures, thermal degradation, water uptake and swelling), and cytotoxicity in vitro was assessed with a MTT test. Moreover, the interaction between the materials and cultured human corneal epithelial cells was studied. The copolymers may be useful for temporary closure of corneal perforations.

Histological and biochemical evaluation of perichondrial transplants in human articular cartilage defects.

From 1986 to 1992, 88 patients with articular defects in the knee were treated with a perichondrial arthroplasty. In this study, we report on the results for 22 biopsies of grafted tissue with a mean follow-up of 21 months. Biopsies were obtained at routine arthroscopy after approximately 1 year or at arthroscopy or arthrotomy at a later stage when patients were operated on again because of recurrent complaints. Biopsies were taken only when a partial failure was present or when there was a clear failure resulting in fibrocartilage, a loose flap, or a loose body. The biopsies were analyzed histologically, biochemically for the amount of type-II collagen, and immunohistochemically with antibodies for types I, II, and X collagen. The well-being of the patients was investigated with use of the Hospital for Special Surgery knee score. The biopsies from 6 patients contained more than 50% hyaline cartilage. At arthroscopy, the mean relative amount of type-II collagen was 56% in the biopsies classified as good. The cartilage of the grafted area was macroscopically normal for eight of the 22 biopsies. Histological and biochemical analysis of biopsies from failed transplants showed fibrocartilage with mainly type-I collagen. These tissues were retrieved primarily from patients with additional abnormalities in the knee joint. It was concluded that adult human perichondrium is able to form hyaline-like cartilage in an isolated cartilage defect in an otherwise healthy knee.

The polymer Polyactive as a bone-filling substance: an experimental study in rabbits.

The biocompatible, osteoconductive and resorbable polymer Polyactive (PA) was investigated for its performance as a bone-graft substitute. The model consisted of a 4 mm borehole, 1.5 cm distal of the major trochanter in both femurs of a rabbit, of which one was filled with a cylinder of porous PA. The other was left untreated. PA70/30 and PA60/40 were investigated, both before and after being incubated with allogenic bone marrow. Analyses were performed after 4, 8, 26 and 52 weeks and comprised dual energy X-ray absorptiometry (DXA) and image analysis of histological sections. DXA revealed an increased bone mineral density in the filled defects compared to the controls, both at the defect and immediately proximal and distal of the defect. Histology showed that gap-bridging had occurred within 8 weeks, with 80%-90% of the pores of PA70/30 and PA60/40 occupied by new bone, and an intimate bone-PA contact. PA70/30 seemed to be more suitable compared to PA60/40, in that the highest amount of bone was formed within the shortest period of time. Incubation of PA with allogenic bone marrow resulted in inflammatory reactions at the sites of implantation, which delayed bone growth, but did not prevent it. It was concluded that PA70/30 and PA60/40 are suitable bone-graft substitutes.

Quantitative histological analysis of bony ingrowth within the biomaterial Polyactive implanted in different bone locations: an experimental study in rabbits.

The quantity of bone formed in cylinders of a newly developed erodible copolymer, Polyactive (PA60/40) was examined. PA60/40 was implanted in three different bone locations in the rabbit: in the cortex, in bone marrow and in trabecular subchondral bone. Bony ingrowth was assessed after 4, 8, 26 and 52 w after the operation and investigated by histology and image analysis. The ingrowth of bone was observed in PA60/40 placed in the cortex from 4 w onwards. After 8 w, more than 90% of the pores of the biomaterial were filled with dense bone. In bone marrow, initially some bone formation was seen. After 26 w, all newly formed bone was resorbed. Subchondral bone formation was less than in the cortex of the femur, but somewhat comparable to the amount of bone found in healthy trabecular bone. Bone formation appeared not to be affected by the degradation of the biomaterial. It was concluded that Polyactive is a suitable bone graft substitute. Bone formation within PA60/40 is site-dependent and this follows Wolff 's law.

A novel method to examine the phenotype of chondrocytes.

Tissue engineering of articular cartilage in order to restore the function of degenerated, diarthrodial joints is currently widely under investigation. The results obtained thus far indicate that proper control of the differentiation of the cells used for this purpose is essential to produce and maintain a hyaline-like matrix. In this study, a procedure is described by which differentiation of chondrocytes in vitro and ex vivo can be studied. The method involves quantitative assessment of mRNA for different collagens, which are markers for differentiation of chondrocytes, by competitive PCR. In a culture system employing human osteoarthritic chondrocytes, mRNAs for the alpha1-chains of collagen types I, II and X are quantified. The procedure is fast, specific and sensitive. However, several controls should be included to ascertain the reliability of the assessment.

Long-term results of rib perichondrial grafts for repair of cartilage defects in the human knee.

Eighty-eight patients with articular cartilage defects in the knee were treated by perichondrial arthroplasty between 1986 and 1992. An autogenous strip of costal perichondrium was fixed in place with fibrin glue, followed by immobilisation, continuous passive motion, and partial weightbearing. The results were evaluated using the Hospital for Special Surgery Score for knee function, radiographs, arthroscopy and the patient's subjective opinion. The results after a mean follow-up of 52 months were good in 38%, fair in 8% and poor in 55%. Previous drilling or shaving of a defect, concomitant osteoarthritis, older age and a long history of complaints proved to be contraindications. Good results were seen in 91% of isolated defects. Perichondrial arthroplasty can be beneficial in the repair of cartilage defects. It will reduce symptoms in carefully selected cases, and avoid more extensive operations for osteoarthritis.

Meniscal repair by fibrocartilage in the dog: characterization of the repair tissue and the role of vascularity.

Lesions in the avascular part of 20 canine menisci were repaired by implantation of a porous polyurethane. Seven menisci were not repaired and served as controls. The repair tissue was characterized by biochemical and immunological analysis. The role of vascularity in healing was studied by perfusion of menisci with Indian ink. Histologically, repair tissue inside the implants initially consisted of fibrous tissue containing type I collagen. After 2 months, fibrocartilaginous tissue developed inside the implants, whereas control defects only showed repair with fibrous tissue. Both type I and type II collagen, the two major collagen types of normal meniscal fibrocartilage, could be detected in this newly formed fibrocartilage. The implant guided vascular tissue from the periphery towards the lesion resulting in healing of the tear. After fibrocartilage had formed, vascularity decreased and was completely absent in mature fibrocartilage. Control defects remained filled with vascular connective tissue. Two-thirds of the longitudinal lesions were found to be healed partially or completely. It is concluded that implantation of a porous polymer does enhance vascularity sufficiently to result in healing of meniscal lesions extending into the avascular part. Healing takes place by repair tissue strongly resembling normal meniscal fibrocartilage.

The effect in vitro of irrigating solutions on intact rat articular cartilage.

Rat patellae were preincubated with culture medium M199 for one hour and then with either fresh culture medium or Ringer's solution, Ringer lactate, Ringer glucose, normal saline or Betadine for another hour. The rate of proteoglycan synthesis in the articular cartilage was then measured by uptake of 35SO4 for the next 16 hours. Cartilage metabolism was inhibited by all of the solutions even after a recovery time of 16 hours. The inhibition was by 5% for Ringer's solution, 10% for Ringer glucose (p < 0.01), 20% for saline and Ringer lactate (p < 0.001) and 55% for Betadine (p < 0.001). Ringer's solution is therefore the best choice for joint irrigation during arthroscopy or other procedures.

Thionin staining of paraffin and plastic embedded sections of cartilage.

The usefulness of thionin for staining cartilage sections embedded in glycol methacrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin O stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin O, thionin did not show metachromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.

Meniscal repair by fibrocartilage? An experimental study in the dog.

Longitudinal lesions in the avascular part of the dog's meniscus were repaired by implantation of a porous polyurethane. Ingrowing repair tissue was characterized by biochemical and immunological analysis. Histologically, repair tissue initially was composed of fibrous tissue containing type I collagen. After 3 months, fibrocartilaginous tissue developed inside the implants, whereas control defects only showed fibrous repair tissue. Both type I and II collagen, the major collagen types of normal meniscal fibrocartilage, could be detected in this newly formed fibrocartilage. It is concluded that fibrocartilage resembling normal meniscal tissue is formed and that longitudinal lesions can be healed after meniscal repair by implantation of a porous polymer.