Luca Valbonetti

Synthetic bone substitute engineered with amniotic epithelial cells enhances bone regeneration after maxillary sinus augmentation.

Background Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined. Aim In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study. Material And Methods Two blocks of synthetic bone substitute (∼0.14 cm3), alone or engineered with 1×106 ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses. Results And Conclusions The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bone-related protein (osteocalcin, OCN) when transplanted into host tissues.

Stemness characteristics and osteogenic potential of sheep amniotic epithelial cells.

We set out to characterize stemness properties and osteogenic potential of sheep AEC (amniotic epithelial cells). AEC were isolated from 3‐month‐old fetuses and expanded in vitro for 12 passages. The morphology, surface markers, stemness markers and osteogenic differentiation were inspected after 1, 6 and 12 passages of expansion, with an average doubling time of 24 h. AEC clearly expressed the stemness markers Oct‐3/4 (octamer‐binding protein‐3/4), Nanog, Sox2 and TERT (telomerase reverse transcriptase) and displayed low levels of global DNA methylation. Culture had moderate effects on cell conditions; some adhesion molecules progressively disappeared from the cell surface, and the expression of Sox2 and TERT was slightly reduced while Nanog increased. No changes occurred in the levels of DNA methylation. Cells organized in 3D spheroids were used for IVD (in vitro differentiation). Within these structures the cells developed a complex intercellular organization that involved extensive intercellular coupling despite continuous cell migration. Marked deposition of calcein in the ECM (extracellular matrix), increased ALP (alkaline phosphatase) activity, expression of bone‐related genes (osteocalcin) and the matrix mineralization shown by Alizarin Red staining demonstrate that AEC can undergo rapid and extensive osteogenic differentiation. AEC introduced in experimental bone lesions survived in the site of implantation for 45 days and supported consistent bone neoformation, thus showing promising potential applications in osteogenic regenerative medicine.

Achilles Tendon Regeneration can be Improved by Amniotic Epithelial Cell Allotransplantation

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14-, CD31-, CD45-, CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial–temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-β1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.

Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

In Vivo Behavior of a Custom-Made 3D Synthetic Bone Substitute in Sinus Augmentation Procedures in Sheep.

In this study, the in vivo behavior of a custom-made three-dimensional (3D) synthetic bone substitute was evaluated when used as scaffold for sinus augmentation procedures in an animal model. The scaffold was a calcium phosphate ceramic fabricated by the direct rapid prototyping technique, dispense-plotting. The geometrical and chemical properties of the scaffold were first analyzed through light and electron scanning microscopes, helium picnometer, and semi-quantitative X-ray diffraction measurements. Then, 6 sheep underwent monolateral sinus augmentation with the fabricated scaffolds. The animals were euthanized after healing periods of 45 and 90 days, and block sections including the grafted area were obtained. Bone samples were subjected to micro computerized tomography, morphological and histomorphometric analyses. A complete integration of the scaffold was reported, with abundant deposition of newly formed bone tissue within the biomaterial pores. Moreover, initial foci of bone remodeling were mainly localized at the periphery of the implanted area after 45 days, while continuous bridges of mature lamellar bone were recorded in 90-day specimens. This evidence supports the hypothesis that bone regeneration proceeds from the periphery to the center of the sinus cavity. These results showed how a technique allowing control of porosity, pore design, and external shape of a ceramic bone substitute may be valuable for producing synthetic bone grafts with good clinical performances.

Postpartum reproductive activities and gestation length in Martina Franca jennies, an endangered Italian donkey breed

The donkey reproductive physiology is still partially known despite the increasing risk of extinction involving several breeds. The present study was designed to describe the postpartum (PP) reproductive performance of an Italian endangered breed: the Martina Franca donkey. To this aim, 52 jennies were monitored to define the foal-heat (FH) and the first and second PP estrus episodes (1st PPe and 2nd PPe). The data indicate that jennies spontaneously recovered reproduction in approximately 10 days after delivery. Then heats occur with a regular interval of approximately 23 days. Estrus length was 1 week in FH and the 2nd PPe and significantly shorter in the 1st PPe. Estrus-ovulation, and delivery-ovulation interval and follicle growth were similar in all animals tested. Pregnancy rate (PR) was lower when natural mating occurred during the FH and 2nd PPe (approximately 60%) than during the 1st PPe (approximately 70%; P < 0.01). In addition, the higher PR (>80%; P < 0.01) was recorded in jennies when the FH occurred after the first week PP and it dropped (<50%) in early FH animals. The PR was also affected by the season and by age: it significantly declined during the autumn-winter season and in subjects older than the sixth year of age. For the first time, the reproductive performance of PP donkeys were defined on a large number of Martina Franca jennies thus offering useful information to improve farm management with an immediate benefit to increase livestock production. This aspect of management improvement might be particularly important if applied to an endangered breed such as Martina Franca donkeys.

Soft Tissue Augmentation of the Face With Autologous Platelet-Derived Growth Factors and Tricalcium Phosphate. Microtomography Evaluation of Mice.

Background:The platelets used in oral, maxillofacial, and plastic surgery are generally grouped as concentrated platelet-rich plasma. The general principle of production consists of a centrifugation, making it possible to eliminate red blood cells, then acellular plasma, to preserve only the concentrated platelets. Objective:The aim of the present study was that micro porous tricalcium phosphate (&bgr;-TCP) mixed with autologous platelet-derived growth factors could be an alternative to fat and hyaluronic acid which are widely used for oral and maxillofacial soft tissue augmentation. Methods:Ten female, 6 to 8-week-old black-haired mice were selected. On 1 cheek was injected the gel of tricalcium phosphate/autologous platelet-derived growth factors, while on the other cheek, was left empty and was used as control. The animals were killed after 8 weeks. Investigator evaluation was based on microtomography observation and comparison of control and test. Results:The microtomography technique demonstrated amorphous radiopaque images projected in the soft tissue parts of each paramedian region of the right cheek, in those sites corresponding to the injection of the gel of tricalcium phosphate/autologous platelet-derived growth factor. Eight weeks after surgery, &bgr;-TCP granules were clearly visible with most remaining within the cheek. The margins of the &bgr;-TCP granules were clear and not diffused within the vicinity of the tissues. Conclusion:The results indicate that micro &bgr;-tricalcium phosphate Ca3(PO4)2 mixed with autologous platelet-derived growth factors material was able to create a lasting three-dimensional soft tissue augmentation and is a promising biomaterial for soft tissue augmentation as a scaffold for cells.

Implant-Abutment Contact Surfaces and Microgap Measurements of Different Implant Connections under 3-Dimensional X-Ray Microtomography

Purpose:The presence of a microgap between implant and abutment could produce a bacterial reservoir which could interfere with the long-term health of the periimplant tissues. The aim of this article was to evaluate, by x-ray 3-dimensional microtomography, implant-abutment contact surfaces and microgaps at the implant-abutment interface in different types of implant-abutment connections. Materials and Methods:A total of 40 implants were used in this in vitro study. Ten implants presented a screw-retained internal hexagon abutment (group I), 10 had a Morse Cone taper internal connection (group II), 10 another type of Morse Cone taper internal connection (group III), and 10 had a screwed trilobed connection (group IV). Results:In both types of Morse Cone internal connections, there was no detectable separation at the implant-abutment in the area of the conical connection, and there was an absolute congruity without any microgaps between abutment and implant. No line was visible separating the implant and the abutment. On the contrary, in the screwed abutment implants, numerous gaps and voids were present. Conclusions:The results of this study support the hypothesis that different types of implant-abutment joints are responsible for the observed differences in bacterial penetration.

Monorchidism in an appaloosa stallion.

THE term ‘monorchidism’ has been used to describe the absence of a descended testicle but should, more strictly, be used to describe the complete absence of one testicle (Parks and others 1989, Santschi and others 1989, Cox 1993, Strong and others 1997). This condition may result from unilateral testicular agenesis or may follow a unilateral vascular accident. ‘Cryptorchidism’ is the correct term to define the retention of a testicle that does not descend into the scrotum (Parker and others 1997). The presurgical evaluation and surgical approach to cryptorchid and monorchid horses are complex diagnostic and surgical challenges (Schumacher 1992). A definitive diagnosis can only be made after surgical exploration of the abdomen, removal of the other testis and then hormonal testing. The human chorionic gonadotropin (hCG) stimulation test in horses over 18 months of age is the most reliable indicator for the presence of vital testicular tissue (Silberzahn and others 1989, Searle and others 1999). This short communication describes the evaluation and treatment of a case of monorchidism in an appaloosa stallion. An eight-year-old appaloosa stallion was referred to the Department of Veterinary Clinical Sciences of the University of Teramo for castration, with no history of previous surgery. Clinical examination showed the absence of the left testicle in the scrotal or inguinal regions. Laparoscopy in dorsal recumbency and under general anaesthesia was planned to diagnose left abdominal cryptorchidism or monorchidism. The horse was fasted for 36 hours and water was withdrawn 12 hours before surgery. Preoperative intravenous flunixin meglumine (1·1 mg/kg), 20,000 iu/kg bodyweight intramuscular penicillin and 6 mg/kg intravenous gentamicin were administered. Premedication with 0·01 mg/kg acepromazine intramuscularly 30 minutes before induction was followed by general anaesthesia induced with 1·1 mg/kg xylazine intravenously and 2·2 mg/kg ketamine intravenously. Anaesthesia was maintained with isoflurane in oxygen. Laparascopic examination according to the technique of Fischer (2002) did not identify the left testicle or vaginal ring, but a cylindrical structure, approximately 1 cm wide, was seen ending near the left internal inguinal ring. It was connected to the peritoneal surface by a short fibrous band. A piece of this structure was collected for histopathological examination. A left parainguinal laparotomy was then performed for inspection of the left dorsal and sublumbar abdominal region, but no testicle was found. Thereafter the right testicle was removed. Histopathology showed the sample from the cylindrical structure to be dense fibrous tissue with no signs of testicular tissue or scarring, suggesting the infarction of a testis. Postoperative therapy included tetanus prophylaxis, and 20,000 iu/kg bodyweight penicillin G given intramuscularly every 12 hours, 6 mg/kg gentamicin administered intramuscularly every 24 hours, and 1·1 mg/kg flunixin meglumine administered intravenously every 24 hours for three days. Eight days after surgery, a hCG stimulation test was performed to determine whether functional testicular tissue was present. This test was repeated 60 days later. Blood samples were collected for determination of plasma testosterone concentration before and one and 24 hours after intravenous administration of 10,000 iu hCG (Searle and others 1997). The blood was stored in sterile vacutainers containing lithium heparin (Venoject; Terumo), and plasma was obtained after centrifugation for 10 minutes at 1200 g. The plasma was then put into Eppendorf tubes at 20°C. A testosterone assay was performed with a radioimmunological assay technique using a commercial kit (Direck RIA; Sorin Biomedica) with an analytical sensitivity of 0·02 ng/ml (Arighi and Bosu 1989, Strong and others 1997). Table 1 shows the values of plasma testosterone before and after hCG administration. Following the testosterone results monorchidism was definitively diagnosed, and six months after surgery the horse showed no stallion-like behaviour. The tubular, fibrous structure detected during laparoscopy may have been the distal part of the undifferentiated ductus deferens, or, more likely, the gubernaculum testis in a stallion with testicular agenesis. It was decided to use a hCG stimulation test rather than the simple measurement of conjugated oestrogens as suggested by Schumacher (1992) to avoid the possibility of false positive results. The basal hormonal values for oestrogens and androgens would be expected to be high in the stallion soon after castration. However, according to Silberzahn and others (1989), the production of oestrogen is almost unresponsive to hCG treatment, and it was therefore decided to use the stimulation test to evaluate the changes in the androgen levels. The first hCG stimulation test was performed eight days after the orchiectomy, as suggested by Santschi and others (1989) and Strong and others (1997). The basal value of the testosterone was considered relatively high, despite the decrease seen one and 24 hours after the administration of hCG. The high basal value during the first test may have been due to the short time after orchiectomy. However, eight weeks after castration, the basal level of androgens was low, and it is suggested that at least this time should be allowed between surgery and testing to achieve the maximum accuracy of any hormonal test. As previously mentioned, a definitive diagnosis of monorchidism in the horse can be achieved only after a complex procedure. Moreover, laparoscopic examination of the abdomen in dorsal recumbency cannot be exhaustive because, even using the Trendelenburg position, dorsal abdominal structures cannot be thoroughly inspected (Galuppo 2002). After removal of the descended testicle, the only reliable way to make a diagnosis is to determine the blood testosterone level and before one and 24 hours after stimulation with 10,000 iu hCG (Arighi and Bosu 1989). In horses with no vital testicular tissue, there should be no increase above basal testosterone concentrations (Searle and others 1997).

Bone Response to Four Dental Implants with Different Surface Topographies: A Histologic and Histometric Study in Minipigs.

This study evaluated four implant surfaces in a minipig model: (1) Kohno Straight dual-engineered surface (DES) (Sweden & Martina); (2) SLActive (Straumann); (3) SM Biotite-H coated with Brushite (DIO); and (4) UF hybrid sandblasted and acid etched (HAS) (DIO). The surfaces presented different topographic features on the macro-, micro-, and nanoscales. After 12 weeks in vivo, significant differences were observed in bone-to-implant contact. UF HAS, presenting moderate microroughness and high nanoroughness, showed some advantage compared to nanorough SM Biotite-H and SLActive. A more pronounced difference was observed between UF HAS and Kohno Straight DES, characterized by a nanosmooth surface. Newly formed bone was observed around all surfaces.