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This study investigated the cotransplantation of bone marrow mesenchymal stromal cells (BMSC) and human umbilical cord endothelial cells (HUVEC), and evaluated their contribution to vascular and bone tissue engineering in vivo. To evaluate the success of osteogenic differentiation and timely vascularization of different osteoconductive scaffolds in vivo, we transferred BMSC and HUVEC pre-cultivated calcium carbonate (CaCO3) and hydroxylapatite (HA) matrices into immunocompromised RNU-rats, and analyzed mineralization, expression of osteopontin, and vascular integration via new vessel formation. After in vivo transplantation, pre-cultivated scaffolds demonstrated overall improved mineralization of 44% for CaCO3 (p = 0.01, SD ± 14.3) and 34% for HA (p = 0.001, SD ± 17.8), as well as improved vascularization of 5.6 vessels/0.1 mm2 on CaCO3 (p There is an increasing demand for improved bone regeneration in tissue engineering. Cotransplantation of mesenchymal stromal cells and endothelial cells often demonstrates synergistic improvements in vitro. However, the benefits or superiority of cotransplantation was not evident in vivo and so will require further investigation.

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper inside into the mechanisms that prevent limb regeneration in humans and other mammals. With regard to tissue engineering and potential medical treatments, such studies may possibly even show ways how to overcome these limitations. First of all, we investigated the regenerative capacity of long-term cultivated cell populations derived from deer antler tissue. The results showed that primary antler cell cultures can be kept alive for more than 27 months without visible signs of apoptosis. Without artifi cial scaffolds, long-term cultures begin to form complex structures which give the impression that antlerogenic cells per se have the ability to build up a “trabecular” structure resembling a spongiosa. Traditionally, antler regeneration has been characterized as a processUniversity Medical Center Goettingen (UMG), Research Group Experimental Osteology, Department of Maxillofacial Surgery, Robert-Koch-Str. 40, D-37075 Goettingen, Germany. aEmail: hrolf@uni-goettingen.de bEmail: wiese@med.uni-goettingen.de *Corresponding authorsList of abbreviation after the text.

Deer antlers are the only mammalian bone structures which regenerate completely every year. Once developed, antlers are cleaned of the velvet-like skin. Presently it is believed that due to velvet shedding the blood supply is interrupted in the solidifying antler bone. Histological examinations were made on different parts of fallow deer antlers investigated from the time of velvet shedding till the antler casting. The present study on hard (polished) antlers revealed living bone with regions presenting living osteocytes, active osteoblasts, osteoid seams and even early stages of trabecular microcallus formation, thus indicating to a continuous bone remodeling. A well developed vascular system was found despite the presence of hard antler bone. The pedicle bone exhibits a rich supply of capillaries and vessels connected to the spongy core of the main branch and the compact bone as well. There is evidence that hard fallow deer antlers possess a functioning vascular system that "keeps the antler moist" resulting in a high impact resistance when fights are most frequent. As late as 3 weeks prior to antler casting a large number of living cells were discovered within the antler core. As we have no doubt that parts of the polished fallow deer antler represent a living bone, we have concluded that a sufficient blood supply of the antler core is maintained almost till the time of antler casting by vessels passing through the antler base.

To investigate the photoperiodic influence on androgen, testis volume, neck girth, and antler-cycles, five fallow bucks were exposed consecutively to different accelerated photoperiodic cycles (three 6-month, three 4-month, and one 3-month cycle). All parameters immediately followed artificial light cycles. Unlike in natural conditions, antler casting, regrowth, and velvet shedding occurred at decreasing day-lengths. The experiment provided further evidence that in fallow bucks the reproductive system is under strictly photoperiodic control and directly and/or indirectly dictates the course of the antler cycle via gonadal hormones. Depending on the test-system, the experiment revealed similar threshold values for the effect of testosterone (T) and/or 5-α-dihydrotestosterone (DHT) concentrations on the antler cycle as previously detected in fallow bucks living under natural conditions. Antlers were cast when T levels dropped below 3 ng/ml, and velvet shedding occurred after T and/or DHT values reached at least 5 to 7 ng/ml. In all cases, amplitudes of androgens remained lower than observed under natural conditions. Frequently during early antler growth, DHT values appeared higher than T levels. With increasing acceleration of the photoperiod, the antler cycle achieved limits of adaptation. The faster the photoperiods were accelerated, the smaller the antlers were developed. In addition, the antler tips were porous, blunt, and not completely mineralized. In all animals, accelerations of the photoperiods evoked phase displacements between measured parameters, pointing to an increasing internal desynchronisation. The present results indicate that the whole reproductive system of fallow deer is directly controlled by the photoperiod and that related morphological and physiological processes are indirectly determined by photoperiodic changes.

Aims: The aim of the present report was to evaluate current methods of in vitro analysis of three-dimensional (3D) scaffolds seeded with human bone marrow stromal cells (hBMSCs) from six bone marrow aspirates for tissue engineered growth of bone. Methods: A series of experiments was conducted to compare methods of cell expansion and to validate analysis of proliferation and differentiation of hBMSCs in long term cultures of up to 40 days in 3D scaffolds of calcium carbonate (CaCO3) and mineralized collagen. Proliferation within the seeded scaffolds was monitored using cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), neutral red (NR) and DNA fluorescence assays and compared with empty controls. Differentiation was assessed by means of ELISA for osteocalcin (OC) and real time PCR for OC and collagen I (Coll I). Results: The results showed that the scaffold differed in seeding efficacy (CaCO3: 53.3%, min. Coll.: 83.3%). The precise identification of the number of cells in biomaterials by MTT, NR and DNA assays was problematic, as MTT and NR assay overestimated the number of cells, whereas DNA assay grossly underestimated the number of cells on the scaffolds. Monitoring of changes over time may be biased by unspecific material-dependent background activity that has to be taken into account. Identification of osteogenic differentiation is not reliable by identifying osteogenic markers such as OC in the supernatant but has to be done on the transcriptional level. Conclusions: It is concluded that monitoring of in vitro procedures for the construction of biohybrid scaffolds requires more emphasis in order to make the cell based approach a reliable treatment option in tissue engineering.

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1+ cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process. peerReviewed

1. 1. Seasonal levels of testosterone (T) and 5-α-DHT (DHT) were determined by RIA in serum of seven adult male fallow deer living under outdoor conditions (Gottingen, FRG; 51.32° NL). 2. 2. The study, spread over six consecutive years, reveals the following seasonal T changes: Minimum concentrations in November or December, around antler casting and at early antler growing phase; increasing levels in the course of late antler development (mineralization) with relatively high values around velvet shedding; peak levels occurring during onset of rutting period followed by a sharp decrease after the rut; and distinct and oscillating T peaks during late winter and spring. 3. 3. For the first time DHT levels were measured in male cervidae. Surprisingly, large values of DHT were found and occasionally levels were higher than T concentrations, but without a significant annual periodicity. 4. 4. In some individuals maximum DHT values were found shortly before and during the rut. 5. 5. During several months of the annual cycle individual T and DHT profiles showed opposite relationships, particularly during antler growing phase and shortly before antler casting. 6. 6. Around rut the size of T peak levels depends on animal age.

Mesenchymal stem cells positive to surface antigen STRO-1 were isolated from regenerating antlers of red deer (Cervus elaphus) and fallow deer (Dama dama) using a magnetic-activated cell sorting (MACS) method. In this study we analysed factors potentially affecting the number of STRO-1+ cells in the cell cultures. With regard to the STRO-1 antigen, we evaluated data from 188 MACS separation procedures of cell cultures cultivated in Dulbecco’s Modified Eagle Medium and 10% fetal calf serum of four fallow deer males (130 procedures) and four red deer males (58 procedures). The analysed factors were the sampling site of the antler or the pedicle, cell passage and type of the cell culture (mixed or STRO-1 negative cell cultures). The percentage of obtained STRO-1+ cells varied greatly from 0.4 to 38.9% for fallow deer and from 1.8 to 16.5% for red deer. We have not found any significant influence of the sampling site. The passage and the type of culture were significant factors for both fallow and red deer cells. The highest numbers of STRO-1+ cells were obtained from the second passage from both fallow and red deer cell cultures (24.6 and 5.5%, respectively). Our experiment revealed that we can maximise the number of STRO-1+ cells in the cultures by manipulating the cultivation factors.