[Mechanical Testing of Tendons Sutured with Newly Developed Biomaterial]. / Mechanické testování slach po suture nove vyvinutým biomateriálem.

PURPOSE OF THE STUDY Tendon injuries continue to be a highly topical issue. Research and clinical activities in this area aim to achieve an optimal repair of the damaged tendon. Such suture is characterised by maximum tensile strength, resistance to gapping at the repair site, preservation of smooth surface, prevention of adhesions and facilitation of fast rehabilitation and active tendon movement. The suture as such is required to show mechanical resistance in particular. Considered optimal is the use of core suture of the tendon in combination with epitendinous suture. The group of researchers has for several years already been exploring new materials. They can contribute to better balance between adequate mechanical strength of the suture and biological support of healing. MATERIAL AND METHODS The study was carried out as an ex vivo experiment on porcine tendon models. A tendon segment was obtained from slaughtered animals and a total rupture of the tendon was imitated by sharp cutting of its central portion. Subsequently, the tendon was repaired by Adelaide suture using coated braided polyester (Ethibond) and two types of new polyamide 6 based (PA6) sutures. The first suture was designed as an unabsorbable polyester core (PES silk) surrounded by absorbable PA6 nanofibres. The second suture was created by braiding a PES silk yarn and two viscose yarns with PA6 nanofibres into a composite surgical suture. As a part of the study also examined was the tensile strength of suture with the use of other stitches, effect of the shape of the needle s point on the tensile strength of the suture and the effect of secured mattress peritendinous suture. The tensile strength of the suture was tested until failure and the achieved maximum load was monitored. RESULTS The PES core yarn with PA6 nanofibre braiding showed lower tensile strength (28.5 ± 5.2 N) than the yarn braided from one PES yarn and two viscose yarns with PA6 nanofibres (45.7 ± 6.7 N). Both newly developed sutures, however, fail to achieve the tensile strength of Ethibond (100.3 ± 19.1 N). In case of Ethibond suture using various types of stitches, the lowest tensile strength was observed in McLarney 4-strand core suture (68.8 ± 18.7 N). A higher tensile strength was achieved by Adelaide 4-strand core suture (83.6 ± 11.2 N). The highest tensile strength was seen in 6-strand core Savage suture (147.4 ± 22.7 N). When the effect of the type of needle was tested, a statistically significant difference between the taper point needle (72.0 ± 7.0 N) and reverse cutting needle (63.3 ± 9.6 N) was observed. In case of McLarney suture the epitendinous stitch increased the tensile strength by 46.2% and in case of Adelaide suture by 48.3%. CONCLUSIONS For tendon core suture, the use of sutures with multiple longitudinal segments seems more appropriate. The epitendinous suture can considerably reinforce the basic load-bearing core suture. Also observed was not an insignificant effect of the needle profile on the resulting tensile strength of the suture. In materials developed by us, more suitable seems to be the design of braiding of absorbable nanofibers with a load-bearing non-absorbable yarn. While the mechanical tensile strength of new materials is lower, the benefits are expected in the form of biological support of healing. Moreover, the nanofibers can be used as a carrier of biological and therapeutic substances. Further improvement of mechanical properties of the newly developed biomaterial can be foreseen if the material of the load-bearing non-absorbable yarn is changed or the load-bearing yarn and nanofibres ratio modified. This pilot study shall use the findings for further development and modification of new materials in basic research and shall also verify the biological aspects and the course of healing in in vivo studies. Key words: tendon, suture, pig, biomaterials, nanofibres, mechanical testing, healing, polyester, Adelaide.

[Use of the Peptigel with Nanofibres in the Bone Defects Healing]. / Vyuzití peptigelu s nanovlákny k lécbe defektu kostní tkáne.

INTRODUCTION Traumatic bone injuries or pathological processes may sometimes result in very extensive bone defects. Currently, the standard procedure applied in clinical humane as well as veterinary medicine to fill a bone defect is the autogenous bone graft which, however, necessitates a more invasive procedure for the patient and in the cases of extensive defects it fails to provide adequate amount of graft. Synthetic bone replacements can be used with no further burden for the patient and can simultaneously be used as the carriers for bioactive molecules or therapeutic drugs. For clinical use, an easy and simple application is one of the requirements that have to be taken into consideration. These requirements are best satisfied by preparations in the form of gel, which may be injected into the defects of various shapes even through minimal surgical approach. MATERIAL AND METHODS Synthetic transparent PGD-AlphaProA hydro-peptide-gel was used as a basis to develop a composite hydrogel scaffold. This gel was enriched by cryogenically ground poly- -caprolactone nanofibers (PCL) in a ratio of 1 ml of gel to 16 µg of nanofibres. In experimental animals (laboratory rat Wistar, n=20), a single regular circular defect of 1.5 mm in diameter was drilled by a low speed drill machine across the whole width of distal femur diaphysis, identically in both the hind legs. In the right hindleg, this defect was filled by injection of 0.05 ml of the composite peptide gel with nanofibers (experimental defect). In the contralateral limb a similar defect was left untreated, without filling (control defect), for spontaneous healing. The group of experimental animals was subsequently divided into four sub-groups (A, B, C, D) for the purpose of further follow-up. One week after the surgical implantation, in the first group of experimental animals (Group A; n = 5) lege artis euthanasia was performed, a radiological examination of both the hind legs was carried out and a sample of the bone from both the control and experimental defect was collected for histologic examination. The other groups of experimental animals were evaluated similarly at 2, 4 and 6 weeks after the surgical procedure (Group B, C, D; n = 5). These groups of experimental animals were assessed using various histological techniques by two independent pathologists. RESULTS A difference between the control and the experimental bone defect was observed only at the healing stage at two weeks after the implantation, when a tendency for greater formation of new bone trabeculas was seen in the defect treated with the composite hydro-peptide-gel with PCL nanofibers. The results show a slightly higher angiogenesis and cellularity at the bone defect site with an increase of newly formed bone tissue and faster colonisation of lamellar bone structures by bone marrow cells at early stages of the healing process (1-2 weeks old defect). In the experimental and control groups, at the later stage of healing (4-6 weeks old defect), the process of healing and bone modelling at the defect site shows no detectable morphological differences. CONCLUSIONS The experimental use of hydro-peptide-gel with PCL nanofibers in vivo in laboratory rats shows very good applicability into the defect site and, compared to the untreated defect within two weeks after the implantation, accelerates the bone healing. This fact could be an advantage especially at the early stage of healing, and thus accelerate the healing of more extensive defects. Key words: peptide gel, polycaprolactone, PCL, replacement, bone, healing, scaffold, nanofibers, biomaterial.

[Comparison between the Range of Movement Canine Real Cervical Spine and Numerical Simulation - Computer Model Validation]. / Porovnání rozsahu pohybu reálné krcní pátere psa s pocítacovou simulací ­ validace pocítacového modelu.

PURPOSE OF THE STUDY In developing new or modifying the existing surgical treatment methods of spine conditions an integral part of ex vivo experiments is the assessment of mechanical, kinematic and dynamic properties of created constructions. The aim of the study is to create an appropriately validated numerical model of canine cervical spine in order to obtain a tool for basic research to be applied in cervical spine surgeries. For this purpose, canine is a suitable model due to the occurrence of similar cervical spine conditions in some breeds of dogs and in humans. The obtained model can also be used in research and in clinical veterinary practice. MATERIAL AND METHODS In order to create a 3D spine model, the LightSpeed 16 (GE, Milwaukee, USA) multidetector computed tomography was used to scan the cervical spine of Doberman Pinscher. The data were transmitted to Mimics 12 software (Materialise HQ, Belgium), in which the individual vertebrae were segmented on CT scans by thresholding. The vertebral geometry was exported to Rhinoceros software (McNeel North America, USA) for modelling, and subsequently the specialised software Abaqus (Dassault Systemes, France) was used to analyse the response of the physiological spine model to external load by the finite element method (FEM). All the FEM based numerical simulations were considered as nonlinear contact statistic tasks. In FEM analyses, angles between individual spinal segments were monitored in dependence on ventroflexion/ /dorziflexion. The data were validated using the latero-lateral radiographs of cervical spine of large breed dogs with no evident clinical signs of cervical spine conditions. The radiographs within the cervical spine range of motion were taken at three different positions: in neutral position, in maximal ventroflexion and in maximal dorziflexion. On X-rays, vertebral inclination angles in monitored spine positions were measured and compared with the results obtain0ed from FEM analyses of the numerical model. RESULTS It is obvious from the results that the physiological spine model tested by the finite element method shows a very similar mechanical behaviour as the physiological canine spine. The biggest difference identified between the resulting values was reported in C6-C7 segment in dorsiflexion (Δφ = 5.95%), or in C4-C5 segment in ventroflexion (Δφ = -3.09%). CONCLUSIONS The comparisons between the mobility of cervical spine in ventroflexion/dorsiflexion on radiographs of the real models and the simulated numerical model by finite element method showed a high degree of results conformity with a minimal difference. Therefore, for future experiments the validated numerical model can be used as a tool of basic research on condition that the results of analyses carried out by finite element method will be affected only by an insignificant error. The computer model, on the other hand, is merely a simplified system and in comparison with the real situation cannot fully evaluate the dynamics of the action of forces in time, their variability, and also the individual effects of supportive skeletal tissues. Based on what has been said above, it is obvious that there is a need to exercise restraint in interpreting the obtained results. Key words: cervical spine, kinematics, numerical modelling, finite element method, canine.

[Ex Vivo Testing of Mechanical Properties of Canine Metacarpal/Metatarsal Bones after Simulated Implant Removal]. / Ex vivo testování mechanických vlastností metakarpálních/metatarzálních kostí psu po simulovaném vyjmutí implantátu.

UNLABELLED: PURPOSE OF THE STUDY In a long-term perspective, it is better to remove implants after fracture healing. However, subsequent full or excessive loading of an extremity may result in refracture, and the bone with holes after screw removal may present a site with predilection for this. The aim of the study was to find ways of how to decrease risk factors for refracture in such a case. This involved support to the mechanical properties of a bone during its remodelling until defects following implant removal are repaired, using a material tolerated by bone tissue and easy to apply. It also included an assessment of the mechanical properties of a bone after filling the holes in it with a newly developed biodegradable polymer-composite gel ("bone paste"). The composite also has a prospect of being used to repair bony defects produced by pathological processes. MATERIAL AND METHODS Experiments were carried out on intact weight-bearing small bones in dogs. A total of 27 specimens of metacarpal/metatarsal bones were used for ex vivo testing. They were divided into three groups: K1 (n = 9) control undamaged bones; K2 (n = 9) control bones with iatrogenic damage simulating holes left after cortical screw removal; EXP (n = 9) experimental specimens in which simulated holes in bone were filled with the biodegradable self-hardening composite. The bone specimens were subjected to three-point bending in the caudocranial direction by a force acting parallel to the direction of drilling in their middiaphyses. The value of maximum load achieved (N) and the corresponding value of a vertical displacement (mm) were recorded in each specimen, then compared and statistically evaluated. RESULTS On application of a maximum load (N), all bone specimens broke in the mid-part of their diaphyses. In group K1 the average maximum force of 595.6 ± 79.5 N was needed to break the bone; in group K2 it was 347.6 ± 58.6 N; and in group EXP it was 458.3 ± 102.7 N. The groups with damaged bones, K2 and EXP, were compared and the difference was found to be statistically significant (p ≤ 0.05). CONCLUSIONS The recently developed biodegradable polymer-composite gel is easy and quick to apply to any defect, regardless of its shape, in bone tissue. The ex vivo mechanical tests on canine short bones showed that the composite applied to defects, which simulated holes left after screw removal, provided sufficient mechanical support to the bone architecture. The results of measuring maximum loading forces were statistically significant. However, before the composite could be recommended for use in veterinary or human medical practice, thorough pre-clinical studies will be required. KEY WORDS: fracture fixation, mechanical testing, bone plate, cortical screw, refracture.

[Intra-articular reinforcement of a partially torn anterior cruciate ligament (ACL) using newly developed UHMWPE biomaterial in combination with Hexalon ACL/PCL screws: ex-vivo mechanical testing of an animal knee model]. / Intraartikulární augmentace parciální ruptury predního zkrízeného vazu (ACL) nove vyvinutým UHMWPE biomateriálem v kombinaci s Hexalon ACL/PCL srouby: ex vivo mechanické testování animálního modelu kolenního kloubu.

PURPOSE OF THE STUDY Recent trends in the experimental surgical management of a partial anterior cruciate ligament (ACL) rupture in animals show repair of an ACL lesion using novel biomaterials both for biomechanical reinforcement of a partially unstable knee and as suitable scaffolds for bone marrow stem cell therapy in a partial ACL tear. The study deals with mechanical testing of the newly developed ultra-high-molecular-weight polyethylene (UHMWPE) biomaterial anchored to bone with Hexalon biodegradable ACL/PCL screws, as a new possibility of intra-articular reinforcement of a partial ACL tear. MATERIAL AND METHODS Two groups of ex vivo pig knee models were prepared and tested as follows: the model of an ACL tear stabilised with UHMWPE biomaterial using a Hexalon ACL/PCL screw (group 1; n = 10) and the model of an ACL tear stabilised with the traditional, and in veterinary medicine used, extracapsular technique involving a monofilament nylon fibre, a clamp and a Securos bone anchor (group 2; n = 11). The models were loaded at a standing angle of 100° and the maximum load (N) and shift (mm) values were recorded. RESULTS In group 1 the average maximal peak force was 167.6 ± 21.7 N and the shift was on average 19.0 ± 4.0 mm. In all 10 specimens, the maximum load made the UHMWPE implant break close to its fixation to the femur but the construct/fixation never failed at the site where the material was anchored to the bone. In group 2, the average maximal peak force was 207.3 ± 49.2 N and the shift was on average 24.1 ± 9.5 mm. The Securos stabilisation failed by pullout of the anchor from the femoral bone in nine out of 11 cases; the monofilament fibre ruptured in two cases. CONCLUSIONS It can be concluded that a UHMWPE substitute used in ex-vivo pig knee models has mechanical properties comparable with clinically used extracapsular Securos stabilisation and, because of its potential to carry stem cells and bioactive substances, it can meet the requirements for an implant appropriate to the unique technique of protecting a partial ACL tear. In addition, it has no critical point of ACL substitute failure at the site of its anchoring to the bone (compared to the previously used PET/PCL substitute). Key words: knee stabilisation, stifle surgery, ultra-high-molecular-weight polyethylene, UHMWPE, nylon monofilament thread, biodegradable screw, bone anchor.

Quality of newly formed cartilaginous tissue in defects of articular surface after transplantation of mesenchymal stem cells in a composite scaffold based on collagen I with chitosan micro- and nanofibres.

The aim of this study was to evaluate macroscopically, histologically and immunohistochemically the quality of newly formed tissue in iatrogenic defects of articular cartilage of the femur condyle in miniature pigs treated with the clinically used method of microfractures in comparison with the transplantation of a combination of a composite scaffold with allogeneic mesenchymal stem cells (MSCs) or the composite scaffold alone. The newly formed cartilaginous tissue filling the defects of articular cartilage after transplantation of the scaffold with MSCs (Group A) had in 60 % of cases a macroscopically smooth surface. In all lesions after the transplantation of the scaffold alone (Group B) or after the method of microfractures (Group C), erosions/fissures or osteophytes were found on the surface. The results of histological and immunohistochemical examination using the modified scoring system according to O'Driscoll were as follows: 14.7+/-3.82 points after transplantations of the scaffold with MSCs (Group A); 5.3+/-2.88 points after transplantations of the scaffold alone (Group B); and 5.2+/-0.64 points after treatment with microfractures (Group C). The O'Driscoll score in animals of Group A was significantly higher than in animals of Group B or Group C (p<0.0005 both). No significant difference was found in the O'Driscoll score between Groups B and C. The treatment of iatrogenic lesions of the articular cartilage surface on the condyles of femur in miniature pigs using transplantation of MSCs in the composite scaffold led to the filling of defects by a tissue of the appearance of hyaline cartilage. Lesions treated by implantation of the scaffold alone or by the method of microfractures were filled with fibrous cartilage with worse macroscopic, histological and immunohistochemical indicators.

Use of allogenic stem cells for the prevention of bone bridge formation in miniature pigs.

This study appears from an experiment previously carried out in New Zealand white rabbits. Allogenic mesenchymal stem cells (MSCs) were transplanted into an iatrogenically-created defect in the lateral section of the distal physis of the left femur in 10 miniature pigs. The right femur with the same defect served as a control. To transfer MSCs, a freshly prepared porous scaffold was used, based on collagen and chitosan, constituting a compact tube into which MSCs were implanted. The pigs were euthanized four months after the transplantation. On average, the left femur with transplanted MSCs grew more in length (0.56+/-0.14 cm) compared with right femurs with physeal defect without transplanted MSCs (0.14+/-0.3 cm). The average angular (valgus) deformity of the left femur had an angle point of 0.78 degrees , following measurement and X-ray examination, whereas in the right femur without transplantation it was 3.7 degrees. The initial results indicate that preventive transplantation of MSCs into a physeal defect may prevent valgus deformity formation and probably also reduce disorders of the longitudinal bone growth. This part of our experiment is significant in the effort to advance MSCs application in human medicine by using pig as a model, which is the next step after experimenting on rabbits.

[New options for management of posttraumatic articular cartilage defects]. / Nové moznosti v lécbe poúrazových defektu kloubní chrupavky.

Articular cartilage trauma, in particular due to its poor healing potential remains a complicated problem in both the adult and paediatric traumatology and orthopedics. In older patients, total endoprosthesis of the joint is a method of choice, however, in younger patients, the situation remains more complicated. In case of osteochondral lesions (arthrosis, chondral fractures. osteochoodrosis dissecns) the ideal management should result in complete recovery of the hyaline cartilage on the traumatized joint surface. Contemporary medicine uses some therapeutic procedures resulting in partial recovery of the articular cartilage structure at the lesion site and several techniques of excisionining the articular surface's injured part and of transplantations of biological grafts. Regarding the above first approach, abrasive methods (micro fractures, small drill holes), which are expected to result in recovery of the articular cartilage through progenitor cells that migrate from the bone marrow to the defect site following subchondral fracturing. In case the injury is managed early, the osteochondral fragment may be fixed and the articular congruence be recovered. Mosaicoplasty using osteochondral auto grafts or other autologous grafts, or more recently using transplantations of autologous chond rocytes, which seem to have a major potential in the hyaline cartilage healing process. However, methodology of the transplant retention at the defect site remains a problem. Furthermore, the use of mesenchymal stem cells, so far in the experimental phase, appears prospective. Pivotal articular cartilage treatment research activities have progressed to a level of searching for a suitable scaffold of perfect qualities. This is the task for cooperation with bioengineering. requiring provision of the most exact differentiation protocol for hyline cartilage producing mesenchymal stem cells (MSCs).

Use of 3D geometry modeling of osteochondrosis-like iatrogenic lesions as a template for press-and-fit scaffold seeded with mesenchymal stem cells.

Computed tomography (CT) is an effective diagnostic modality for three-dimensional imaging of bone structures, including the geometry of their defects. The aim of the study was to create and optimize 3D geometrical and real plastic models of the distal femoral component of the knee with joint surface defects. Input data included CT images of stifle joints in twenty miniature pigs with iatrogenic osteochondrosis-like lesions in medial femoral condyle of the left knee. The animals were examined eight and sixteen weeks after surgery. Philips MX 8000 MX and View workstation were used for scanning parallel plane cross section slices and Cartesian discrete volume creation. On the average, 100 slices were performed in each stifle joint. Slice matrices size was 512 x 512 with slice thickness of 1 mm. Pixel (voxel) size in the slice plane was 0.5 mm (with average accuracy of +/-0.5 mm and typical volume size 512 x 512 x 100 voxels). Three-dimensional processing of CT data and 3D geometrical modelling, using interactive computer graphic system MediTools formerly developed here, consisted of tissue segmentation (raster based method combination and 5 % of manual correction), vectorization by the marching-cubes method, smoothing and decimation. Stifle- joint CT images of three individuals of different body size (small, medium and large) were selected to make the real plastic models of their distal femurs from plaster composite using rapid prototyping technology of Zcorporation. Accuracy of the modeling was +/- 0.5 mm. The real plastic models of distal femurs can be used as a template for developing custom made press and fit scaffold implants seeded with mesenchymal stem cells that might be subsequently implanted into iatrogenic joint surface defects for articular cartilage-repair enhancement.