Spinning around or stagnation - what do osteoblasts and chondroblasts really like?

OBJECTIVE: The influence of cytomechanical forces in cellular migration, proliferation and differentiation of mesenchymal stem cells (MSCs) is still poorly understood in detail. METHODS: Human MSCs were isolated and cultivated onto the surface of a 3 x 3 mm porcine collagen I / III carrier. After incubation, cell cultures were transferred to the different cultures systems: regular static tissue flasks (group I), spinner flasks (group II) and rotating wall vessels (group III). Following standard protocols cells were stimulated lineage specific towards the osteogenic and chondrogenic lines. To evaluate the effects of applied cytomechanical forces towards cellular differentiation distinct parameters were measured (morphology, antigen and antigen expression) after a total cultivation period of 21 days in vitro. RESULTS: Depending on the cultivation technique we found significant differences in both gen and protein expression. CONCLUSION: Cytomechanical forces with rotational components strongly influence the osteogenic and chondrogenic differentiation.

Extensive H(+) release by bone substitutes affects biocompatibility in vitro testing.

Bone substitutes are widespread in orthopedic and trauma surgery to restore critical bony defects and/or promote local bone healing. Cell culture systems have been used for many years to screen biomaterials for their toxicity and biocompatibility. This study applies a human bone marrow cell culture system to evaluate the toxic in vitro effects of soluble components of different bone substitutes, which are already in clinical use. Different specimens of tricalcium phosphates (TCP) (Vitoss, Cerasorb), nondecalcified bovine bone (Lubboc), demineralized human bone matrices (DBM) (Grafton Flex/Putty), and collagen I/III matrix (ACI-Maix) were tested in Dulbecco's modified Eagle's medium (DMEM) and MesenCult culture solution and compared with a biomaterial-free cell culture. Biocompatibility parameters were cell viability evaluated by phase-contrast microscopy and laser flow cytometry, morphology, and the local H(+) release by bone substitutes. There were significant differences (p < 0.05) between the tested biomaterials and culture solutions. Collagen I/III, non-demineralized bovine bone, and TCP materials showed advantages for cell survival over other tested biomaterials (average values of vital cells/mL MesenCult/DMEM: Collagen I/III: 1090/1083; Vitoss: 893/483; Cerasorb: 471/523; Lubboc: 815/410; Grafton Putty: 61/44; Grafton Flex: 149/57). Especially the DBM materials lead to a significant decrease of pH, which is considered to be a major factor for cell death. DMEM culture solution supports cell survival for those bone substitutes that induce an alkaline reaction, whereas MesenCult media promotes cell vitality in biomaterials, which leads to an acidification of culture solution.

The critical size bony defect in a small animal for bone healing studies (I): Comparative anatomical study on rats' femur.

Laboratory rats are small animal models which are often used for scientific investigations in medicine. So far there are only few scientific data about the meaning of these small animal models for in vivo bone healing studies available in literature. Although the rat's femur with its cyclic loadings during gait is an appropriate model for investigations in the field of experimental orthopaedics and traumatology there is a lack of morphometric information with respect to its anatomy. The aim of this study is to evaluate the anatomy of rat femurs in two species, which are often performed in animal experimental medicine. These morphometric data should contribute to develope an appropriated osseous fragment fixation system in the rat's femur. The femurs of Wistar (WR) and Sprague Dawley (SDR) cadavers were prepared and analysed by x-rays in two standard planes. The results were compared with the corresponding data for humans by literature. It could be demonstrated that SDR showed a higher caput-collum-diaphyseal and antetorsion angle, but a lower transcondylar femur valgus angle compared to WR. Cortical thickness, bone marrow cavity diameter and femur length were higher in WR. Wistar rat's femur anatomy shows more similarities to human anatomy than Sprague Dawley rats.

The critical size bony defect in a small animal for bone healing studies (II): implant evolution and surgical technique on a rat's femur.

In the preclinical field of orthopaedic and trauma surgery critical size bony defects (CDS) were used to evaluate the biocompatibility and allow to investigate the osteoinductivity and -conductivity of bone substitutes. Concerning the anatomical size the laboratory rat indicates a lower limit in small animals which are appropriate for experiments on bone. The aim of this study was to define a CSD, to develop a suitable fixation system to stabilize bony fragments in CSD and to point out the specialities of the surgical technique. These informations should help for to design and practice studies concerning bone healing on rat's femur. Based on previously acquired anatomical data of rat's femur, the technical challenges and anatomical specialities of different osteosynthesis techniques in rat's femur surgery are demonstrated. Our experiences with different fixation systems and techniques lead to the development of an external fixator, which guarantees for a stable bone fragment fixation, prevents severe soft tissue damage, allows of a roentgenologic evaluation of the defect zone and prevents from undesired direct biomaterial-implant interactions. Neither the proximal nor the distal femoral nailing technique is appropriate for a stable fixation in CSD of rat's femur. To evaluate the reliability of an own developed external fixator 42 nude rats with a 4.0 mm CSD were investigated clinically and roentgenologically over 10 weeks. The external fixator showed only a small implant failure rate. A solid fusion of the bone fragments was not observed within the 10 weeks follow-up period.

Developmental organization of neurophysin neurons in the human brain.

Neurophysin (NPH) was detected immunohistochemically in 34 human brains ranging in age from 10 weeks of gestation (wg) to 3 months postnatal. Weakly-stained NPH-immunoreactive (NPH-IR) cells were already aggregated in the lateral hypothalamus in the supraoptic nucleus at 10 wg, the first time point examined. From this time, there was a clear and consistent chronology in the first appearance of NPH-immunoreactivity in different cell groups progressing from the supraoptic nucleus at 10 wg to cells in the accessory NPH cell group at 13 wg, paraventricular nucleus at 14 wg, suprachiasmatic nucleus at 18 wg and various other well defined clusters in the basal forebrain at 18-20 wg. NPH-IR fibers were present in the hypothalamo-hypophyseal tract from 10 wg, and together with other available evidence, our findings suggest the presence of a potentially functional hypothalamohypophyseal system by the end of the first trimester. NPH staining patterns and orientations of cells suggest that NPH-IR cells originate from the region of the hypothalamic sulcus in a manner consistent with animal studies, and migrate to their settling areas before expressing NPH-immunoreactivity. In spite of the likelihood that most NPH-IR cells (with the probable exception of those in the suprachiasmatic nucleus) derive from a single primordium, the final organization of NPH-IR cells consists of many scattered groups, as seen in the late fetal period and mature brain. Developmental analysis provides further evidence that there is a high degree of conservation in the topographic organization of the numerous diverse NPH-IR cell groups in humans and other mammals, suggesting that the separation and organization of these groups may be of functional importance.

Organisation and maturation of the human thalamus as revealed by CD15.

The distribution of the CD15 antigen (CD15, 3-fucosyl-N-acetyl-lactosamine, Lewis x) has been studied immunohistochemically in the fetal human thalamus. Its changing patterns could be related to three successive, but overlapping, periods primarily due to its association with radial glial cells, neuropil, and neural cell bodies, respectively. From 9 weeks of gestation (wg), a subset of CD15-positive radial glial cells distinguished the neuroepithelium of the ventral thalamus, a characteristic also seen in the developing mouse. Distal processes of the radial glial cells converged at the root of the forebrain choroid tenia, which was also CD15 positive. From 13 wg until approximately 20 wg, CD15-positive neuropil labeling marked the differentiation areas of prospective nuclei within the dorsal thalamus and progressively outlined their territories in a time sequence, which appeared specific for each nucleus. CD15 labeling of differentiating nuclei of the ventral, medial, anterior, and intralaminar thalamic divisions showed a transient topographic relationship with restricted areas of the ventricular wall. After 26 wg, CD15 immunoreactivity was observed in subpopulations of glial cells and neurons. Transient CD15 immunoreactivity was also found in delimited compartments within the subventricular region. The time of CD15 expression, its location, and cellular association suggest that CD15 is involved in segmentation of diencephalon, in the specification of differentiating nuclear areas and initial processes regarding the formation of intercellular contacts and cellular maturation.

Spatiotemporal expression gradients of the carbohydrate antigen (CD15) (Lewis X) during development of the human basal ganglia.

The developmental expression pattern of the carbohydrate epitope CD15 (Lewis X, Le X) (alpha1-->3-fucosyl-N-acetyl-lactosamine) has been immunocytochemically evaluated in paraffin sections within the human basal ganglia from 10 weeks gestation to three years after birth. At 11 weeks of gestation, CD15 (Le X) positive radial glial cells were located in the anterior and dorsal parts of the lateral ganglionic eminence. Their processes ran from the subventricular zone radially in a highly ordered fashion to the dorsolateral margin of the caudate nucleus and further to the lateral rim of the putamen. At 12 weeks of gestation, strands of CD15 (Le X) material continued to the pial surface, forming a continuous CD15 (Le X) positive borderline separating the accumbens nucleus and olfactory tubercle from the piriform cortex. At 13 weeks of gestation the dorsal putamen was completely CD15 (Le X) immunoreactive along its perimeter and CD15 (Le X) patches, consisting of fine granular material, appeared at the dorsolateral margin of the putamen at this age; while the first CD15 (Le X) patches in the caudate nucleus were observed four weeks later. The matrix compartment of the caudate and dorsal putamen became gradually stained by granular CD15 (Le X) positive material into which CD15 (Le X) immunoreactive somata were embedded. The striking contrast in staining between patch and matrix compartments disappeared shortly after birth. The ventral striatum did not become immunoreactive until the last few weeks before birth. After the formation of CD15 (Le X) positive patches in the striatum (from 12 weeks of gestation), delicate CD15 (Le X) fibres, often accumulated in bundles and related to the striatal patches, became apparent coursing towards the external pallidal lamina and the globus pallidus. Immunoreactivity in the globus pallidus itself was transient, emerging from 16 weeks of gestation, reaching a peak at 21 weeks of gestation and disappearing by birth. Both processes, i.e. the occurrence of CD15 (Le X) striatopallidal fibres and the emerging immunoreactivity in their pallidal target, may be interrelated, so that ingrowing CD15 (Le X) positive axons from the striatum provoke CD15 (Le X) expression in the external and internal pallidum. The variable patterns and intensities of CD15 (Le X) expression are possibly related to periods of maturation of the striatum and the establishment of functional interactions within the basal ganglia. Differential staining of patch and matrix in the developing neostriatum suggests that a distinct phase of cellular adhesion or dishesion mediated by the CD15 (Le X) epitope occurs during establishment of the patch and matrix regions.

Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells.

Mesenchymal progenitor cells derived from cord blood (unrestringated somatic stem cells, USSC) and bone marrow (mesenchymal stem cells, MSC) are able to differentiate under defined culture conditions into at least bone, cartilage, adipose and muscle cells in vitro. The culture media and other in vitro conditions influence the osteogenic differentiation potency of both cell types. To increase and expand the number of osteoblasts in vitro an optimization of culture conditions is required. The aim of this study was to evaluate different culture media toward their osteogenic promoting capacity on human USSCs and MSCs in vitro. Immunohistochemical stainings against osteonectin (ON), osteopontin (OP) served as markers for an osteoblastic differentiation. Cellular morphology was analysed by light microscopy technique. We found significant differences between bone marrow and cord blood derived stem cells towards an osteoblastic differentiation. Considering the number of osteoblasts MesenCult seems to have advantages in bone marrow progenitor cells, whereas low glucose DMEM and HAMS-F12 promoted an osteoblastic differentiation in cord blood derived cells more than other tested media.

Osteoblastic potency of bone marrow cells cultivated on functionalized biometals with cyclic RGD-peptide.

The fixation of cementless endoprostheses requires excellent fixation at the bone implant interface. Although the surface structures of these implants are designed to promote osteoblastic differentiation, poor bone quality may prevent or delay osseointegration. There is evidence that RGD peptides known as recognition motifs for various integrins, promote cellular adhesion, influence cellular proliferation, and differentiation of local cells. In this study, five different metal surfaces were analyzed: Sandblasted (TiSa) and polished (TiPol) Ti6Al4V, porocoated (CCPor) and polished (CCPol) cobalt chrome and polished stainless steel (SS) were coated by ethanol amine and poly(ethylene glycol) to attach covalently RGD peptides. Human mesenchymal stromal cells of healthy donors were cultivated onto prior functionalized metal surfaces for 14 days without osteogenic stimulation. Cell proliferation and differentiation were quantitatively evaluated for native (I), NaOH pre-activated (II), NaOH pre-activated, and PEG-coated (III) as well as for RGD (IV) coated surfaces. The RGD immobilization efficiency was analyzed by epi-fluorescence spectroscopy, cell morphology was documented by light and scanning electron microscopy. The RGD-binding efficiency was TiSa > TiPol > SS > CCPor > CCPol. RGD coated surfaces showed the highest average cell proliferation on CCPol > SS > CCPor > TiSa ≥ TiPol, whereas cellular differentiation mostly correlated with the observed proliferation results, such as CCPol > TiSa > SS > CCPor > TiPol. Considering statistical analyses (significance level of α = 0.05), the RGD-coating of all biometals in comparison and in respect of their particular controls showed no significant improvement in cellular proliferation and osteoblastic differentiation.