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The Hippo pathway is an evolutionarily conserved pathway crucial for regulating tissue size and for limiting cancer development. However, recent work has also uncovered key roles for the mammalian Hippo kinases, Mst1/2, in driving appropriate immune responses by directing T cell migration, morphology, survival, differentiation, and activation. In this review, we discuss the classical signaling pathways orchestrated by the Hippo signaling pathway, and describe how Mst1/2 direct T cell function by mechanisms not seeming to involve the classical Hippo pathway. We also discuss why Mst1/2 might have different functions within organ systems and the immune system. Overall, understanding how Mst1/2 transmit signals to discrete biological processes in different cell types might allow for the development of better drug therapies for the treatments of cancers and immune-related diseases.

Immediate implant placement often presents challenges in terms of predictably obtaining soft-tissue coverage over the implant site. While delayed implant placement offers the ability for soft tissues to grow and invade the extraction socket making their attachment around implants more predictable, immediate implant placement poses a significant risk of bacterial invasion towards the implant surface as a result of insignificant soft-tissue volume. Soft-tissue grafting techniques have often been proposed for use during immediate implant placement to augment soft-tissue deficiencies, including the use of either palatal connective tissue grafts (CTGs) or collagen-derived scaffolds. However, both of these approaches have significant drawbacks in that CTGs are harvested with high patient morbidity and collagen scaffolds remain avascular and acelluar posing a risk of infection/implant contamination. More recently, platelet-rich fibrin (PRF) has been proposed as an economical and biological means to speed soft-tissue wound healing. In combination with immediate implant placement, PRF offers an easily procurable low-cost regenerative modality that offers an efficient way to improve soft-tissue attachment around implants. Furthermore, the supra-physiological concentration of defense-fighting leukocytes in PRF, combined with a dense fibrin meshwork, is known to prevent early bacterial contamination of implant surfaces, and the biological concentrations of autologous growth factors in PRF is known to increase tissue regeneration. This article discusses soft-tissue grafting techniques associated with immediate implant placement, presents several cases demonstrating the use of PRF in routine immediate implant placement, and further discusses the biological and economic advantages of PRF for the management of soft-tissue grafting during immediate implant placement.

This study investigated the possible mechanisms for explaining interanimal variation in efficiency of feed utilization in intact male Holstein calves. Additionally, we examined whether the feed efficiency (FE) ranking of calves (n = 26) changed due to age and/or diet quality. Calves were evaluated during three periods (P1, P2, and P3) while fed a high-quality diet (calculated mobilizable energy [ME] of 11.8 MJ/kg DM) during P1 and P3, and a low-quality diet (calculated ME of 7.7 MJ/kg DM) during P2. The study periods were 84, 119, and 127 d, respectively. Initial ages of the calves in P1, P2, and P3 were 7, 11, and 15 mo, respectively, and initial body weight (BW) were 245, 367, and 458 kg, respectively. Individual dry matter intake (DMI), average daily gain (ADG), diet digestibility, and heat production (HP) were measured in all periods. The measured FE indexes were: residual feed intake (RFI), the gain-to-feed ratio (G:F), residual gain (RG), residual gain and intake (RIG), the ratio of HP-to-ME intake (HP/MEI), and residual heat production (RHP). For statistical analysis, animals' performance data in each period, were ranked by RFI, and categorized into high-, medium-, and low-RFI groups (H-RFI, M-RFI, and L-RFI). RFI was not correlated with in vivo digestibility, age, BW, BCS, or ADG in all three periods. The L-RFI group had lowest DMI, MEI, HP, retained energy (RE), and RE/ADG. Chemical analysis of the longissimus dorsi muscle shows that the L-RFI group had a higher percentage of protein and a lower percentage of fat compared to the H-RFI group. We suggested that the main mechanism separating L- from H-RFI calves is the protein-to-fat ratio in the deposited tissues. When efficiency was related to kg/day (DMI and ADG) and not to daily retained energy, the selected efficient L-RFI calves deposited more protein and less fat per daily gain than less efficient H-RFI calves. However, when the significant greater heat increment and maintenance energy requirement of protein compared to fat deposition in tissue were considered, we could not exclude the hypothesis that variation in efficiency is partly explained by efficient energy utilization. The ranking classification of calves to groups according to their RFI efficiency was independent of diet quality and age.

AIM The local delivery of growth factors via gene therapy has gained tremendous awareness in recent years due to their sustained growth factor delivery to target tissues. The aim of this study was to fabricate and investigate a scaffold able to release growth factors via gene therapy for the repair of periodontal tissues. MATERIALS AND METHODS Novel mesoporous bioglass (MBG)/silk fibrin scaffold combined with BMP7 and/or PDGF-B adenovirus was fabricated and tested in vitro for cell migration, proliferation and differentiation. Furthermore, acute-type buccal dehiscence periodontal defects (mesiodistal width × depth: 5 × 5 mm) were created on the buccal portion of the maxillary premolars in five normal male beagle dogs (12 months old, 15.0 ± 2.0 kg) and histologically examined for periodontal regeneration following implantation of the following five groups: (1) no scaffold, (2) MBG/silk scaffold alone, (3) scaffold + adPDGF-B, (4) scaffold + adBMP7, (5) scaffold + adPDGF-b + adBMP7. RESULTS In vitro findings demonstrated that adPDGF-B was able to rapidly recruit periodontal ligament (PDL) cells over sixfold more effectively than adBMP7, whereas adBMP7 was more able to induce osteoblast differentiation of PDL cells. In vivo findings demonstrate that scaffolds loaded with adPDGF-B were able to partially regenerate the periodontal ligament while adBMP7 scaffolds primarily improved new bone formation. The combination of both adPDGF-B and adBMP7 synergistically promoted periodontal regeneration by allowing up to two times greater regeneration of the periodontal ligament, alveolar bone and cementum when compared to each adenovirus used alone. CONCLUSIONS Although both PDGF-B and BMP7 are individually capable of promoting periodontal regeneration to some degree, their combination synergistically promotes wound healing in acute-type buccal dehiscence periodontal defects when delivered simultaneously. This study demonstrates the promise for successful delivery of low-cost, effective growth factor delivery via gene therapy for the treatment of periodontal defects.

Semaphorins have been recently targeted as new molecules directly implicated in the cell-cell communication that occurs between osteoclasts and osteoblasts. Overexpression of certain semaphorins, such as semaphorin4D (sema4D), is found in an osteoporotic phenotype and plays a key role in osteoclast activity by suppressing osteoblast maturation, thus significantly altering the bone modeling cycle. In the present study, we fabricate a site-specific bone-targeting drug-delivery system from polymeric nanoparticles with the incorporation of siRNA interference molecule for sema4D and demonstrate their cellular uptake and intracellular trafficking within osteoclasts, thus preventing the suppression of osteoblast activity. We then demonstrate in an osteoporotic animal model induced by ovariectomy that weekly intravenous injections led to a significantly greater number of active osteoblasts at the bone surface, resulting in higher bone volume in compromised animals. The findings from the present study demonstrate a novel and promising site-specific therapeutic option for the treatment of osteoporosis via interference of the sema4D-plexin cell communication pathway between osteoclasts and osteoblasts. © 2014 American Society for Bone and Mineral Research.

Recombinant human bone morphogenic protein (rhBMP) 9 has recently been reported to have more osteopromotive potential in vitro when compared to rhBMP2. The aim of the present study was to investigate the bone-inducing potential of rhBMP2 and rhBMP9. We compared rhBMP2, rhBMP7, and rhBMP9 at five different concentrations and showed convincingly that rhBMP9 possesses much greater potential for osteoblast differentiation even at 20 times lower concentrations in vitro. We further show that Noggin, an inhibitor for rhBMP2-induced osteogenesis, did not alter rhBMP9-induced osteogenesis. Thereafter, we show for the first time that rhBMP9 loaded onto atelo-collagen membranes is osteoinductive and has greater potential to form ectopic bone formation when compared to rhBMP2 even at four times lower doses. Similarly new bone formation of rhBMP2 and 9 when loaded on deproteinized bovine bone mineral (DBBM) was investigated in a rabbit calvarial defect. At 8 weeks, both rhBMP2 and rhBMP9 induced significantly higher new bone formation when compared to DBBM alone samples. Interestingly, once again four times lower dose of rhBMP9 group induced comparable or even greater levels of new bone height and new bone area when compared to the rhBMP2 group. The present study revealed that (1) rhBMP9 is capable of inducing ectopic new bone formation in vivo and (2) up to four times lower doses of rhBMP9 may be utilized to regenerate same-size bone defects when compared to rhBMP2. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1561-1574, 2018.

Bone grafts are widely used in bone regeneration to increase the speed and quality of new bone formation. While they are routinely characterized based on their biocompatible and bioactive properties, they also exert a profound impact on host immune responses, which in turn can display a significant effect on the healing and repair process. In this study, we investigated the role of macrophage behavior on deproteinized bovine bone matrix (DBBM, BioOss) to investigate their impact on creating either a pro- or anti-inflammatory microenvironment for tissue integration. RT-PCR and immunofluorescence staining results demonstrated the ability for RAW 264.7 cells to polarize toward M2 wound-healing macrophages in response to DBBM and positive control (IL-4). Interestingly, significantly higher expression of interleukin-10 and higher number of multinucleated giant cells (MNGCs) was observed in the DBBM group. Thereafter, conditioned media (CM) from macrophages cultured with DBBM seeded with MC3T3-E1 cells demonstrated a marked increase in osteoblast differentiation. Noteworthy, this effect was reversed by blocking IL10 with addition of IL10 antibody to CM from the DBBM macrophages. Furthermore, the use of dendritic cell specific transmembrane protein (DC-STAMP)-knockout to inhibit MNGC formation in the DBBM group resulted in a significant reduction in osteoblast differentiation, indication a pivotal role for MNGCs in biomaterials-induced osteogenesis. The results from this study indicate convincingly that the immune response of macrophages towards DBBM has a potent effect on osteoblast differentiation. Furthermore, DBBM promoted macrophage fusion and polarization towards an M2 wound-healing phenotype, further created a microenvironment favoring biomaterial-induced osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1236-1246, 2018.

Platelet rich fibrin (PRF) is a blood concentrate system obtained by centrifugation of peripheral blood. First PRF matrices exhibited solid fibrin scaffold, more recently liquid PRF-based matrix was developed by reducing the relative centrifugation force and time. The aim of this study was to systematically evaluate the influence of RCF (relative centrifugal force) on cell types and growth factor release within injectable PRF- in the range of 60-966 g using consistent centrifugation time. Numbers of cells was analyzed using automated cell counting (platelets, leukocytes, neutrophils, lymphocytes and monocytes) and histomorphometrically (CD 61, CD- 45, CD-15+, CD-68+, CD-3+ and CD-20). ELISA was utilized to quantify the concentration of growth factors and cytokines including PDGF-BB, TGF-β1, EGF, VEGF and MMP-9. Leukocytes, neutrophils, monocytes and lymphocytes had significantly higher total cell numbers using lower RCF. Whereas, platelets in the low and medium RCF ranges both demonstrated significantly higher values when compared to the high RCF group. Histomorphometrical analysis showed a significantly high number of CD61+, CD-45+ and CD-15+ cells in the low RCF group whereas CD-68+, CD-3+ and CD-20+ demonstrated no statistically significant differences between all groups. Total growth factor release of PDGF-BB, TGF-β1 and EGF had similar values using low and medium RCF, which were both significantly higher than those in the high RCF group. VEGF and MMP-9 were significantly higher in the low RCF group compared to high RCF. These findings support the LSCC (low speed centrifugation concept), which confirms that improved PRF-based matrices may be generated through RCF reduction. The enhanced regenerative potential of PRF-based matrices makes them a potential source to serve as a natural drug delivery system. However, further pre-clinical and clinical studies are required to evaluate the regeneration capacity of this system.

Regenerative therapies are commonly needed prior to implant placement in the posterior maxilla following tooth extraction. The aim of this study was to investigate the anatomical features of the atrophic posterior maxilla that might affect surgical approach selection and technique and risk of intra- and postoperative complications. Cone beam computed tomography files were screened to identify pristine atrophic maxillary ridges in need of bone augmentation (ridge height < 10 mm). Ridge height and width and lateral wall thickness (LWT) were measured at different levels, and palatonasal recess and lateral wall morphology were calculated. The influence of site was assessed using generalized estimation equations. The linear correlation between parameters was estimated with Pearson coefficient, and principal component analysis (PCA) was performed to establish patterns of multiple correlations. In the 433 edentulous sites and 6,062 measurements analyzed, the strongest correlations corresponded to LWT measurements at the different levels. Most of the parameters examined presented linearity (P < .001), allowing establishment of four main morphologic patterns according to anatomical features. The anatomical structures of the atrophic posterior maxilla follow linear patterns that might predict complications due to an anatomical feature and thus success in regenerative therapy.

Achieving a long-term esthetic implant restoration in the maxillary anterior area is sometimes unpredictable. This article reviews several risk indicators of implant mucosal recession and loss of interproximal papillae, and how to prevent these risks. Results obtained from the literature suggest that the following factors are strongly related to implant soft-tissue complications: buccally positioned implant placement, a thin gingival biotype, lack of an adequate amount of keratinized mucosa, implant placement after ridge augmentation procedures, an increased distance of tooth bone peak to the contact point, and an inadequate horizontal implant-tooth distance. Factors that have been shown to favor longterm stability and improve implant esthetic outcomes include implant placement in an ideal 3-dimensional position, use of platform-switching implant/abutment designs, and performing soft-tissue grafting. Careful assessment of the above factors and adoption of suggested guidelines are critical to executing successful implant placement with favorable esthetic outcomes while minimizing soft-tissue complications.

OBJECTIVES: Recently, recombinant human bone morphogenetic protein 9 (rhBMP9) has been characterized as one of the most osteogenic growth factors among the 15 human BMPs. The aim of the present study was to investigate the effects of rhBMP9 in comparison to the clinically utilized rhBMP2 on in vitro cell behavior when combined with two bone graft materials including demineralized freeze-dried bone allografts (DFDBAs) and biphasic calcium phosphate (BCP). MATERIALS AND METHODS: The absorption and release kinetics of rhBMPs from DFDBA and BCP were investigated by ELISA. Moreover, murine bone stromal ST2 cell behavior was investigated on DFDBA or BCP seeded on (1) graft only, (2) rhBMP2 (10 ng/ml), (3) rhBMP2 (100 ng/ml), (4) rhBMP9 (10 ng/ml), and (5) rhBMP9 (100 ng/ml). The effects of rhBMPs on DFDBA and BCP were assessed for cell adhesion, proliferation, and osteoblast differentiation by alkaline phosphatase (ALP) activity, alizarin red staining, and real-time PCR for genes encoding Runx2, ALP, and bone sialoprotein (BSP). RESULTS: While both BMPs were gradually released from DFDBA and BCP over time, significantly higher adsorption was observed on BCP when compared to DFDBA. Cell attachment and proliferation was higher on BCP with little influence of either rhBMP2/9. Despite rhBMPs having relatively no effect on cell attachment/proliferation, a pronounced and marked effect was observed on osteoblast differentiation for both rhBMP2/9. Interestingly, it was observed that rhBMP9 induced significantly higher ALP activity, alizarin red staining, and messenger RNA (mRNA) levels of ALP and BSP when compared to rhBMP2. Our results also revealed higher differentiation for rhBMP2/9 with BCP when compared to DFDBA most likely as a result of higher growth factor adsorption. CONCLUSION: While both rhBMP2/9 combined with DFDBA or BCP induced osteoblast differentiation, rhBMP9 induced greater osteoblast differentiation when compared to rhBMP2. CLINICAL RELEVANCE: rhBMP9 may be a recombinant growth factor with higher potential to induce new bone formation when compared to rhBMP2. Further in vivo studies are necessary to characterize its regenerative potential in various animal models. KEYWORDS: BCP; BMP2; BMP9; Biphasic calcium phosphate; Bone morphogenetic proteins; Bone regeneration; Bone tissue engineering; DFDBA; Demineralized freeze-dried bone allografts

Bone morphogenetic protein 9 (BMP9) has previously been characterized as the strongest osteoinductive growth factor among the BMP family. The aim of the present study was to evaluate the possibility of combining rhBMP9 with an injectable biphasic calcium phosphate (I-BCP, maxresorb inject®), since I-BCP is an easy to handle biomaterial with ideal properties for bone augmentation procedures. The adsorption potential of rhBMP9 as well as the cell behavior of bone stromal ST2 cells were investigated on cell viability, adhesion, proliferation and osteogenic differentiation for I-BCP combined with/without rhBMP9 in vitro. I-BCP demonstrated excellent adsorption/retention potential of rhBMP9 with a slow and steady release over a 10 day period by ELISA. Cell attachment at 8 hours and cell proliferation at 1, 3 and 5 days was decreased on I-BCP with/without rhBMP9 when compared to control tissue-culture plastic. While I-BCP had little influence on osteoblast differentiation, its combination with rhBMP9 significantly increased ALP activity at 7 days and mRNA levels of osteoblast differentiation markers including ALP and osteocalcin at 14 days. I-BCP served as an excellent carrier for rhBMP9 clearly demonstrating its osteoinductive potential. We therefore confirm the great potential of rhBMP9 to serve as a future regenerative growth factor for bone applications.

Recent research has demonstrated that recombinant human bone morphogenetic protein 9 (rhBMP9) has been considered the most osteoinductive growth factor of the BMP-family. In the present study, rhBMP9 was investigated for its influence in combination with two biomaterials for bone regenerative medicine. Either porcine-derived collagen membrane (CM) or deproteinized bovine bone mineral (DBM) combined with 20 µg of rhBMP9 were implanted in 6 mm rabbit calvarial defects. Bone augmentation was evaluated by microCT and histomorphometry at 8 weeks post-surgery. Both CM + rhBMP9 and DBM + rhBMP9 groups significantly promoted mineralized tissue volume (microCT) and area, new bone height and area (histomorphometric measurements) when compared to CM and DBM alone groups or control (empty). All specimens in the CM + rhBMP9 group but not all in the DBM + rhBMP9 group induced a complete horizontal bone defect closure. Multinucleated giant cells (MNGCs) were observed directly in contact with DBM surfaces irrespective of rhBMP9, whereas CM was generally not associated to the presence of MNGCs. When combined with rhBMP9, DBM augmented a larger volume of mineralized tissue (including the mineralized bone graft), whereas CM induced greater volume of native host bone. While DBM in combination with rhBMP9 induced higher mineralized tissue mostly associated with the bone grafting material, CM may have presented preferable results based on a higher horizontal defect closure with a faster regeneration of host new bone. The effect of including collagen within the carrier system of rhBMP9 on bone regeneration justifies further evaluation of this combination procedure in larger animal models. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2655-2661, 2017.

Over 15 years have now passed since enamel matrix derivative (EMD) emerged as an agent capable of periodontal regeneration. Following thorough investigation, evidenced-based clinical application is now established for a multitude of clinical settings to promote regeneration of periodontal hard tissues. Despite the large number of studies and review articles written on this topic, no single review has compiled the influence of EMD on tissue inflammation, an area of research that merits substantial attention in periodontology. The aim of the present review was to gather all studies that deal with the effects of EMD on tissue inflammation with particular interest in the cellular mechanisms involved in inflammation and soft tissue wound healing/resolution. The effects of EMD on monocytes, macrophages, lymphocytes, neutrophils, fibroblasts and endothelial cells were investigated for changes in cell behavior as well as release of inflammatory markers, including interleukins, prostaglandins, tumor necrosis factor-α, matrix metalloproteinases and members of the OPG-RANKL pathway. In summary, studies listed in this review have reported that EMD is able to significantly decrease interleukin-1b and RANKL expression, increase prostaglandin E2 and OPG expression, increase proliferation and migration of T lymphocytes, induce monocyte differentiation, increase bacterial and tissue debris clearance, as well as increase fibroplasias and angiogenesis by inducing endothelial cell proliferation, migration and capillary-like sprout formation. The outcomes from the present review article indicate that EMD is able to affect substantially the inflammatory and healing responses and lay the groundwork for future investigation in the field.

Semaphorin 4d (Sema4d) has been proposed as a novel target gene for the treatment of osteoporosis. Recently, we fabricated a site-specific bone-targeting system from polymeric nanoparticles that demonstrates an ability to prevent bone loss in an osteoporotic model by interfering with Sema4d gene expression using small interference RNA (siRNA) molecules. The aim of the present investigation was to determine the effects of this targeting system on the periodontium, an area of high bone turnover. We demonstrated, by single photon emission computed tomography, that intravenous injection of this molecule in ovariectomized Balb/C mice is able to target alveolar bone peaking 4 hr post-injection. We then compared, by histological analysis, the bone volume/total volume (BV/TV), alveolar bone height loss, immunohistochemical expression of Sema4d, and total number of osteoclasts in mandibular alveolar bone. Four treatment modalities were compared as follows: (1) sham-operated, (2) OVX-operated, (3) OVX+estrogen replacement therapy, and (4) OVX+siRNA-Sema4d animals. The results from the present study demonstrate that an osteoporotic condition significantly increases alveolar bone height loss, and that the therapeutic effects via bone-targeting systems featuring interference of Sema4d are able to partly counteract alveolar bone loss caused by osteoporosis. While the future therapeutic demand for the large number of patients suffering from osteoporosis faces many challenges, we demonstrate within the present study an effective drug-delivery moiety with anabolic effects on the bone remodeling cycle able to locate and target alveolar bone regeneration.

The purpose of the present study was to synthesize a new composites scaffold containing poly(γ-benzyl-L-glutamate) modified hydroxyapatite/(poly (L-lactic acid)) (PBLG-g-HA/PLLA) and to investigate their in vitro behaviour on bone mesenchymal stromal cells (BMSCs). The results demonstrated that BMSC proliferation was significantly increased on PBLG-g-HA/PLLA scaffolds after 3 and 7 days post seeding when compared to PLLA and HA/PLLA scaffolds. The in vitro osteogenic differentiation also favoured the composite PBLG-g-HA/PLLA scaffolds when compared to controls by significantly increasing Runx2, ALP or osteocalcin mRNA expression as assessed by real-time PCR. The results illustrate the potential of PBLG-g-HA/ PLLA scaffolds for bone tissue engineering applications. And the in vivo testing further confirms the PBLG-g-HA/PLLA scaffolds’ potentioal for healing critical bone defects.

Thirty-five lactating dairy cows throughout weeks of lactation (WOL) 16 to 30 were used to determine optimal time needed for reliable measurement of performance variables, and to classify the cows into high-, medium-, and low-efficiency groups. Individual performance variables [body weight (BW), dry matter intake (DMI), and milk production] were measured daily with a computerized monitoring system. Body condition was visually scored weekly and used to calculate retained or depleted body energy as a result of fat content change (REF). Milk composition was analyzed weekly. Body weight, DMI, and total recovered energy (RE), which represents energy in milk production plus REF, were summarized weekly. Efficiency was calculated as RE/DMI and as residual feed intake (RFI; i.e., the difference between actual and expected DMI), which was calculated from multiple linear regression of DMI dependence on BW(0.75) and RE. Unexpectedly, it was found that BW did not affect DMI and RE/DMI. Changes and relative changes in phenotypic coefficient of variation and correlations among data from shortened tests ranging from 1 wk (WOL 16) to a sequence of 15-wk tests were used to determine optimal test period durations for 5 traits: BW, DMI, RE, RE/DMI, and RFI. Traits were fitted into a mixed model with repeated measures. For each week, the traits were summarized as a sequence of cumulative data, starting from WOL 16 and cumulated over periods that increased in 1-wk steps up to WOL 16 to 29. Weekly cumulations were compared with those for entire test period (WOL 16 to 30). Consistency of each cow's efficiency classification as high, medium, or low was tested by the total-agreement procedure; the kappa index P-value was used. Throughout WOL 16 to 30, the effects of increasing test period duration on between-animal coefficient of variation differed with respect to the various performance variables and RE/DMI: it tended to change with respect to BW, did not change with respect to DMI, and decreased with respect to RE and RE/DMI. In conclusion, compared with a 15-wk study, a 2-wk study can classify RFI and RE/DMI to 3 efficiency levels, with an individual correlation coefficient of 0.6. When the study was carried out over 3 wk or more, the lowest significant index of the classification was P

BACKGROUND AND OBJECTIVE Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. MATERIAL AND METHODS Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. RESULTS The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. CONCLUSION The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

This study measured the effects of including 5% NaOH-treated corn straw (T-CS) as a substitute for 15% wheat hay in the control total mixed ration (TMR) of lactating cows on performance, digestibility, and rumen microbial profile. Two groups of 21 cows each, similar in initial performance, were fed individually 1 of the 2 TMR examined. Voluntary dry matter intake of cows fed the control TMR was 4.3% higher than that of the T-CS cows, but in vivo dry matter and organic matter digestibilities of both groups were similar. Crude protein digestibility was higher in the control cows but digestibility of neutral detergent fiber polysaccharides (cellulose and hemicelluloses) was higher in the T-CS TMR. This was followed by 4.6% reduction in rumination time of the T-CS group. A slightly higher milk yield was observed in the control cows compared with the T-CS group; however, milk fat and milk protein content were higher in cows fed the T-CS TMR. This was reflected in 1.3% increase in energy-corrected milk yield and 5.34% increase in production efficiency (energy-corrected milk yield/intake) of the T-CS cows compared with the control. Welfare of the cows, as assessed by length of daily recumbence time, was improved by feeding the T-CS TMR relative to the control group. As a whole, the rumen bacterial community was significantly modulated in the T-CS group in the experimental period compared with the preexperimental period, whereas the bacterial community of the control group remained unchanged during this period. Out of the 8 bacterial species that were quantified using real-time PCR, a notable decrease in cellulolytic bacteria was observed in the T-CS group, as well as an increase in lactic acid-utilizing bacteria. These results illustrate the effect of T-CS on the composition of rumen microbiota, which may play a role in improving the performance of the lactating cow.

BACKGROUND: Harvesting techniques can affect cellular parameters of autogenous bone grafts in vitro. Whether these differences translate to in vivo bone formation, however, remains unknown. OBJECTIVE: The purpose of this study was to assess the impact of different harvesting techniques on bone formation and graft resorption in vivo. MATERIAL AND METHODS: Four harvesting techniques were used: (i) corticocancellous blocks particulated by a bone mill; (ii) bone scraper; (iii) piezosurgery; and (iv) bone slurry collected from a filter device upon drilling. The grafts were placed into bone defects in the mandibles of 12 minipigs. The animals were sacrificed after 1, 2, 4 and 8 weeks of healing. Histology and histomorphometrical analyses were performed to assess bone formation and graft resorption. An explorative statistical analysis was performed. RESULTS: The amount of new bone increased, while the amount of residual bone decreased over time with all harvesting techniques. At all given time points, no significant advantage of any harvesting technique on bone formation was observed. The harvesting technique, however, affected bone formation and the amount of residual graft within the overall healing period. Friedman test revealed an impact of the harvesting technique on residual bone graft after 2 and 4 weeks. At the later time point, post hoc testing showed more newly formed bone in association with bone graft processed by bone mill than harvested by bone scraper and piezosurgery. CONCLUSIONS: Transplantation of autogenous bone particles harvested with four techniques in the present model resulted in moderate differences in terms of bone formation and graft resorption.

Background Replacement bone grafting materials are used clinically for a variety of clinical procedures to augment and replace lost or missing bone. Little information is available regarding their degradation properties. Purpose The aim of the present study was to investigate the degradation rate and modes of degradation of four commonly used bone grafting materials. Materials and Methods A natural bone mineral (NBM) of bovine origin, NBM in combination with enamel matrix derivative (EMD), LifeNet demineralized freeze-dried bone allograft (DFDBA), and Osteotech DFDBA were analyzed for particle degradation over time in 3 mm femur defects created in female Wistar rats. At 2, 4, and 8 weeks postimplantation, femur defects were assigned to histological analysis. Hematoxylin and eosin, Safranin O, tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa B ligand (RANKL), and matrix metalloproteinase-2 (MMP-2) staining were performed to determine the rate of particle degradation, number of osteoclasts around particles, and intensity and localization of TRAP, RANKL, and MMP-2 staining. Results In the present study, NBM particles demonstrated little signs of degradation. The combination of NBM with EMD significantly increased the number of osteoclasts around NBM particles and increased expression of RANKL and MMP-2 specifically around particle surface. Only minor resorption was observed. Both DFDBA particles showed much faster degradation of particles. Interestingly, fewer osteoclasts were found on their surface when compared with NBM particles, specifically on Osteotech DFDBA particles, suggesting an alternative mode of degradation. Osteotech DFDBA particles demonstrated significantly faster degradation when compared with all other bone grafts. No obvious increase in TRAP, RANKL, or MMP2 was observed to validate this fast rate of degradation. Conclusions The results from the present study demonstrate a wide range of particle degradation between various commonly commercially available bone grafts. Further research to determine the precise mechanisms that influence particle degradation is necessary.

Tissue engineering generally requires three basic elements; stem/progenitor cells, inductive agents and a biomaterial scaffold; the latter is one of the key components which directly influences cellular activity and matrix formation. Commonly used scaffolds to repair defects in general do not induce stem cell recruitment, which is an essential element to tissue regeneration. In this study, fabrication of a scaffold which is capable of restoring damaged tissue through the recruitment of mesenchymal stem cells (MSCs) by gene therapy of the gene encoding platelet-derived growth factor-B (PDGF-B) was investigated. PDGF-B adenovirus (AdPDGF) was combined into novel mesoporous bioglass-silk fibrin scaffolds, which were characterized for their controlled release and sustained bioactivity. Our results demonstrate that these scaffolds can release PDGF-B adenovirus for up to 3 weeks and increase MSC recruitment, both in vitro and following subcutaneous implantation in mice. Osseous calvarial defects in mice further demonstrate the ability of these scaffolds to enhance tissue regeneration through stem cell homing. This study demonstrates the potent ability of host stem cells to regenerate tissue defects through recruitment of MSCs via gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.

Objective The purpose of this investigation was to compare the osteoinductive and osteopromotive potential of two widely used demineralized freeze-dried bone allografts (DFDBA) (Osteotech® DFDBA and LifeNet® DFDBA). Material and Methods Twenty-seven male Wistar rats (mean body weight 200 g) were treated with either DFDBA from Osteotech and LifeNet or control for femoral and intramuscular defects and assigned to histological analysis at 2, 4, and 8 weeks postimplantation. Hematoxylin and eosin (H&E), Safranin-O, tartrate-resistant acid phosphatase (TRAP), and osteopontin (OPN) staining were performed. Quantitative analysis of mineralized new bone to total volume (BV/TV) was assessed by micro-computed tomography. Results Both allografts demonstrated osteoinductive potential at 2 weeks as assessed by intramuscular bone formation. LifeNet DFDBA displayed continual new bone formation at 4 and 8 weeks, whereas Osteotech particles were fully resorbed by 4 weeks postimplantation. Femur defects demonstrated significantly greater BV/TV at 4 and 8 weeks with higher expression of OPN staining around LifeNet DFDBA particles. TRAP-positive cells were visible in and around both allograft materials. Conclusion The results from the present study indicate that variability among allografts exists. In the present, LifeNet DFDBA supported more new bone formation. Further larger animal models or clinical trials are required to validate these findings.

The most common complication for patients with postmenopausal osteoporosis is bone-related defects and fractures. While routine medication has a high probability of undesirable side effects, new approaches have aimed to develop regeneration procedures that stimulate new bone formation while reversing bone loss. Recently, we have synthesized a new hybrid CaP/silk scaffold with a CaP-phase distribution and pore architecture better suited to facilitate cell differentiation and bone formation. The aim of the present study was to compare the involved remodeling process and therapeutic effect of porous CaP/silk composite scaffolds upon local implantation into osteoporotic defects. Wistar rats were used to induce postmenopausal osteoporotic model by bilateral ovariectomy. The pure silk and hybrid CaP/silk scaffolds were implanted into critical sized defects created in distal femoral epiphysis. After 14 and 28 days, the in vivo osteogenetic efficiency was evaluated by μCT analysis, hematoxylin and eosin staining, Safranin O staining, tartrate-resistant acid phosphatase staining, and immunohistochemical assessment. Animals with or without critical-sized defects were used as drill or blank controls, respectively. The osteoporotic defect model was well established with significantly decreased μCT parameters of BV/TV, Tb.N and increased Tb.Sp, porosity, combined with changes in histological observations. During the healing process, the critical-sized drill control defects failed to regenerate appreciable bone tissue, while more significantly increased bone formation and mineralization with dynamic scaffold degradation and decreased osteoclastic bone resorption could be detected within defects with hybrid CaP/silk scaffolds compared to pure silk scaffolds.

a b s t r a c t This study examined digestibility for lambs and nutritive value for lactating ewes of a total mixed ration (TMR) containing Cephalaria joppensis (CJ) silage as a wheat silage substitute. Yield of CJ forage was 16.4 t dry matter (DM) per ha, which is 37% higher than that of the neighboring wheat. The fresh-cut wheat and CJ were packed separately and wrapped in polyethylene layers to produce bales of silage kept outdoors for 90 days. The CJ and wheat silage bales were opened, combined with alfalfa hay (at a 2:1 ratio, DM basis) and mixed with additional TMR components, repacked and rewrapped to produce TMR bales kept outdoors for 30 days. The packing and wrapping created anaerobic conditions and ensilage occurred within the TMR bales, resulting in a significant reduction in pH of the wheat-TMR (W-TMR) and CJ-TMR from 6.02 and 6.07, respectively, to 4.1 and 4.4, respectively. DM loss in the two types of TMR bales during ensilage was similar. Experiment 1 measured the intake and digestibility of the ensiled W-TMR vs. CJ-TMR by two groups of six Assaf lambs each, fed individually in metabolic cages. Intake and digestibility of DM, crude pro- tein and neutral detergent fiber were similar for the two TMRs tested. However, cellulose digestibility was slightly higher in the W-TMR. Experiment 2 measured milk production and composition in two groups of 45 lactating Assaf ewes each, fed the two ensiled TMRs plus a constant amount of mixed concentrate pellets. The ewes fed the CJ-TMR were char- acterized by similar yields of daily milk (2.26-2.36 kg), protein (0.11 kg), fat (0.15 kg) and 6% fat-corrected milk (3.06-3.08 kg), but slightly higher concentrations of protein, fat and total milk solids compared with sheep fed the W-TMR. Changes in body weight and body condition score were similar in the two groups of ewes. Data show that CJ silage can be included in a TMR for lactating sheep as high-quality forage, with a nutritive value similar to that of wheat silage.

Time-varying linear regression via flexible least squares is used to determine temperature-dependent kinetic parameters during low-pressure, steady-state, temperature- programmed desorption from catalytic surfaces. The flexible least squares approach optimizes time-varying parameters by minimizing dynamic and measurement discrepancies between a linear theoretical model and experimental data using linear regression. The effectiveness of this approach is demonstrated by calculation of accurate temperature-dependent activation energies, preexponential factors, and differential conversion functions for the evolution of 3- methyl-2-oxetanone (β-lactone) during the selective oxidation of isobutane over aluminum phosphomolybdates. C

BACKGROUND Recombinant human bone morphogenetic protein 9 (rhBMP9) has been considered the most osteoinductive growth factor of the BMP-family and has much translation potential for guided bone regeneration (GBR) procedures. PURPOSE The aim of this study was to compare bone formation using rhBMP9 loaded with different carrier systems including deprotenized bovine bone mineral (BioOss, BO) or collagen barrier membranes (BioGide, BG) in a rabbit GBR model. MATERIALS AND METHODS rhBMP9 was loaded either on BO; named BO/BMP9, or BG; named BG/BMP9 to investigate the better carrier system for rhBMP9. New bone formation was quantified in a rabbit calvarial defect model using four groups; (1) control (empty, n = 9), (2) BO + BG (n = 9), (3) BO/BMP9 + BG (n = 9; BMP9 loaded onto BO), and (4) BO + BG/BMP9 (n = 9; BMP9 loaded onto BG) by radiographically and histologically at 8 weeks post-surgery. RESULTS Both BO/BMP9 + BG and BO + BG/BMP9 samples significantly promoted new bone formation when compared to BO + BG samples based on parameters including mineralized tissue volume by microCT analysis, as well as new bone height and new bone area by histomorphometry. Interestingly, BO + BG/BMP9 samples but not BO/BMP9 + BG achieved near perfect horizontal bone defect closure, while demonstrating new bone layers in the defect areas implanted with BG materials and bone formation around BO materials. CONCLUSION Both BO and BG positively induced bone formation with rhBMP9 in an experimental rabbit GBR model when compared to BO + BG alone. This study revealed that BG-loaded with rhBMP9 promoted better wound closure when compared to BO-loaded with rhBMP9. GBR procedures with growth factors may thus benefit from loading rhBMP9 onto BG-collagen barrier membranes when compared to BO-bone grafting particles. Future large animal studies with different types of bone grafts and barrier membranes are needed to further investigate these trends.

Background: Damage or exposure of the dental pulp requires immediate therapeutic intervention. Methods: This study assessed the biocompatibility of a silver-containing PLGA/TCP-nanofabric scaffold (PLGA/Ag-TCP) in two in vitro models, i.e. the material adapted on pre-cultured cells and cells directly cultured on the material, respectively. Collagen saffolds with and without hyaluronan acid (Coll-HA; Coll) using both cell culturing methods and cells growing on culture plates served as reference. Cell viability and proliferation were assessed after 24, 48, and 72 h based on formazan formation and BrdU incorporation. Scaffolds were harvested. Gene expression of interleukin(IL)-6, tumor necrosis factor (TNF)-alpha, and alkaline phosphatase (AP) was assessed 24 h after stimulation. Results: In both models formazan formation and BrdU incorporation was reduced by PLGA/Ag-TCP on dental pulp cells, while no significant reduction was found in cells with Coll and Coll-HA. Cells with PLGA/Ag-TCP for 72 h showed similar relative BrdU incorporation than cells stimulated with Coll and Coll-HA. A prominent increase in the pro-inflammatory genes IL-6 and TNF-α was observed when cells were cultured with PLGA/Ag-TCP compared to the other groups. This increase was parallel with a slight increase in AP expression. Overall, no differences between the two culture methods were observed. Conclusions: PLGA/Ag-TCP decreased viability and proliferation rate of human dental pulp cells and increased the pro-inflammatory capacity and alkaline phosphatase expression. Whether these cellular responses observed in vitro translate into pulp regeneration in vivo will be assessed in further studies., BMC Oral Health, 17, ISSN:1472-6831

To study the clinical effect of short implant placement using osteotome sinus floor elevation technique and tent-pole grafting technique with recombinant human bone morphogenetic protein 2 (rhBMP-2) in severely resorbed maxillary area.Eleven patients with insufficient bone height in the posterior maxillary area were included. According to the native bone height and crown height space (CHS), the patients were divided into two groups: immediate placement of short implants with simultaneous bone augmentation (group A, 5 patients) and delayed dental implant placement (4 to 6 months) after bone augmentation. The rhBMP-2 was added into a deproteinised bovine bone mineral (DBBM) bone grafting material to shorten the treatment procedure and enhance the final effect of bone augmentation in both groups. Tent-pole grafting technique was applied for vertical bone augmentation in group B (6 patients).The success rate of the implants placed was 100% in both groups. In group A, the short implants treatment was successful, with a vertical gain of 1.5 to 6.4 mm in bone height after 4 to 6 months. In group B, the tent-pole grafting procedure in combination with DBBM and rhBMP-2 increased vertical bone height between 3.1 and 8.1 mm, an optimistic and adequate increase for implant placement. This bone increase was maintained following implant placement and final crown placement in the maxillary region (3.5 to 7.3 mm).The tent-pole grafting technique was a viable alternative choice to lateral sinus floor elevation in cases with excessive CHS. The application of rhBMP-2 with a shortened treatment time demonstrated positive outcomes in sinus floor augmentation procedures.

Various strategies have been employed to speed tissue regeneration using bioactive molecules. Interestingly, platelet concentrates derived from a patient’s own blood have been utilized as a regenerative strategy in recent years. In the present study, a novel liquid platelet formulation prepared without the use of anti-coagulants (injectable-platelet-rich fibrin, i-PRF) was compared to standard platelet-rich plasma (PRP) with gingival fibroblasts cultured on smooth and roughened titanium implant surfaces. Standard PRP and i-PRF (centrifuged at 700 rpm (60× g) for 3 min) were compared by assays for fibroblast biocompatibility, migration, adhesion, proliferation, as well as expression of platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), collagen1 (COL1) and fibronectin (FN). The results demonstrate that i-PRF induced significantly higher cell migration, as well as higher messenger RNA (mRNA) levels of PDGF, TGF-β, collagen1 and fibronectin when compared to PRP. Furthermore, collagen1 synthesis was highest in the i-PRF group. These findings demonstrate that liquid platelet concentrates can be formulated without the use of anticoagulants and present much translational potential for future research. Future animal and clinical trials are now necessary to further investigate the potential of utilizing i-PRF for soft tissue regenerative protocols in combination with various biomaterials.

PURPOSE: Collagen barrier membranes were first introduced to regenerative periodontal and oral surgery to prevent fast ingrowing soft tissues (ie, epithelium and connective tissue) into the defect space. More recent attempts have aimed at combining collagen membranes with various biologics/growth factors to speed up the healing process and improve the quality of regenerated tissues. Recently, a new formulation of enamel matrix derivative in a liquid carrier system (Osteogain) has demonstrated improved physico-chemical properties for the adsorption of enamel matrix derivative to facilitate protein adsorption to biomaterials. The aim of this pioneering study was to investigate the use of enamel matrix derivative in a liquid carrier system in combination with collagen barrier membranes for its ability to promote osteoblast cell behavior in vitro. MATERIALS AND METHODS: Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto porcine-derived collagen membranes alone (control) or porcine membranes + enamel matrix derivative in a liquid carrier system. Control and enamel matrix derivative-coated membranes were compared for cell recruitment and cell adhesion at 8 hours; cell proliferation at 1, 3, and 5 days; and real-time polymerase chain reaction (PCR) at 3 and 14 days for genes encoding Runx2, collagen1alpha2, alkaline phosphatase, and bone sialoprotein. Furthermore, alizarin red staining was used to investigate mineralization. RESULTS: A significant increase in cell adhesion was observed at 8 hours for barrier membranes coated with enamel matrix derivative in a liquid carrier system, whereas no significant difference could be observed for cell proliferation or cell recruitment. Enamel matrix derivative in a liquid carrier system significantly increased alkaline phosphatase mRNA levels 2.5-fold and collagen1alpha2 levels 1.7-fold at 3 days, as well as bone sialoprotein levels twofold at 14 days postseeding. Furthermore, collagen membranes coated with enamel matrix derivative in a liquid carrier system demonstrated a sixfold increase in alizarin red staining at 14 days when compared with collagen membrane alone. CONCLUSION: The combination of enamel matrix derivative in a liquid carrier system with a barrier membrane significantly increased cell attachment, differentiation, and mineralization of osteoblasts in vitro. Future animal testing is required to fully characterize the additional benefits of combining enamel matrix derivative in a liquid carrier system with a barrier membrane for guided bone or tissue regeneration.

BACKGROUND Hyaluronic acid (HA) has been reported to have a positive effect on periodontal wound healing following nonsurgical and surgical therapy. However, to date, a few basic in vitro studies have been reported to investigating the potential of HA on human periodontal ligament (PDL) cell regeneration. Therefore, the aim of this study was to investigate the effect of HA on PDL cell compatibility, proliferation, and differentiation in vitro. METHODS Either non-cross-linked (HA_ncl) or cross-linked (HA_cl) HA was investigated. Human PDL cells were seeded in 7 conditions as follows (1) Control tissue culture plastic (TCP) (2) dilution of HA_ncl (1:100), (3) dilution of HA_ncl (1:10), 4) HA_ncl directly coated onto TCP, (5) dilution of HA_cl (1:100), 6) dilution of HA_cl (1:10) and (7) HA_cl directly coated onto TCP. Samples were then investigated for cell viability using a live/dead assay, an inflammatory reaction using real-time PCR and ELISA for MMP2, IL-1 and cell proliferation via an MTS assay. Furthermore, the osteogenic potential of PDL cells was assessed by alkaline phosphatase(ALP) activity, collagen1(COL1) and osteocalcin(OCN) immunostaining, alizarin red staining, and real-time PCR for genes encoding Runx2, COL1, ALP, and OCN. RESULTS Both HA_ncl and HA_cl showed high PDL cell viability (greater than 90%) irrespective of the culturing conditions. Furthermore, no significant difference in both mRNA and protein levels of proinflammatory cytokines, including MMP2 and IL-1 expression was observed. Both diluted HA_ncl and HA_cl significantly increased cell numbers compared to the controlled TCP samples at 3 and 5 days. HA_ncl and HA_cl in standard cell growth media significantly decreased ALP staining, COL1 immunostaining and down-regulated early osteogenic differentiation, including Runx2, COL1, and OCN mRNA levels when compared to control samples. When osteogenic differentiation medium (ODM) was added, interestingly, the expression of early osteogenic markers increased by demonstrating higher levels of COL1 and ALP expression; especially in HA 1:10 diluted condition. Late stage osteogenic markers remained inhibited. CONCLUSIONS Both non-cross-linked and cross-linked HA maintained high PDL cell viability, increased proliferation, and early osteogenic differentiation. However, HA was consistently associated with a significant decrease in late osteogenic differentiation of primary human PDL cells. Future in vitro and animal research is necessary to further characterize the effect of HA on periodontal regeneration.

The immune response to bone biomaterials plays a critical role during early inflammation and biomaterial-induced osteogenesis. Dendritic cells (DCs) possess a significant impact during immune responses to biomaterials. While conventional strategies to evaluate the osteogenic potential of bone substitute materials focus mainly on osteoblast differentiation. Therefore the present study was to investigate the interactions DCs to porous β-tricalcium phosphate (β-TCP) to investigate whether DCs participate in the material-coordinated osteogenic process. DC maturation and function were investigated by detecting the expression of cell surface markers and inflammatory cytokines by RT-PCR. DCs seeded on β-TCP showed a decrease in co-stimulatory molecules (CD86 and CD40) and pro-inflammatory gene IFN-γ but an increase in anti-inflammatory genes including IL-1ra, IL-4, and IL-10, as well as TGF-β1. Thereafter, conditioned media was harvested from DCs seeded on standard tissue culture plastic and β-TCP to be utilized with osteoblast cell cultures. It was found that conditioned media from DCs seeded on β-TCP markedly promoted osteoblast differentiation by increasing the expression of osteogenic genes RUNX2, ALP, OCN as well as increased ALP activity and alizarin red staining in vitro. In conclusion, the findings from the present study point to the potential crucial role of DCs in biomaterial-induced osteogenesis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1703-1711, 2017.

The aim of the present study was to evaluate the effect of injectable platelet-rich fibrin (i-PRF) on cultivated chondrocytes and osteochondral regeneration in critical-sized osteochondral defect of the rabbit’s knee in comparison to autologous platelet-rich plasma (PRP). Chondrocytes were first investigated for their ability to proliferate and differentiate in response to PRP and i-PRF. Thereafter, full-thickness critical-sized osteochondral defects 5 mm in diameter and 5 mm in depth were created in the knee joint of 12 adult female New Zealand White rabbits. Defects were regenerated with either PRP or i-PRF and compared to control. Animals were sacrificed at 4 and 12 weeks postoperatively and evaluated histologically by macroscopic and microscopic examination for cartilage regeneration. i-PRF significantly promoted chondrocyte proliferation and mRNA levels of Sox9, collagen type II, and aggrecan when compared to PRP and control. Histological analysis revealed that at 4 weeks, macroscopic ICRS scores from the i-PRF group were significantly enhanced when compared to the PRP and control groups. At 12 weeks post surgery, the microscopic ICRS scores demonstrated that the i-PRF group significantly improved cartilage regeneration when compared to PRP. In conclusion, the use of i-PRF using the low speed centrifugation concept significantly promoted chondrocyte activity and further improved cartilage regeneration when compared to PRP. The histological results revealed early and better cartilage regeneration within 4 weeks postoperatively when i-PRF was utilized and the results were maintained at 12 weeks. Future clinical studies are now needed investigating the regenerative potential of i-PRF in comparison to PRP for knee regeneration.