This study assessed the regeneration potential of mesenchymal stem cells (MSC) from adipose tissue associated with platelet-rich plasma (PRP) in bone regeneration.

Mucopolysaccharidosis type II (MPS II, OMIM 309900) is an X-linked disorder caused by a deficiency of lysosomal enzyme iduronate-2-sulfatase (IDS). The clinical manifestations of MPS II involve cognitive decline, bone deformity, and visceral disorders. These manifestations are closely associated with IDS enzyme activity, which catalyzes the stepwise degradation of heparan sulfate and dermatan sulfate. In this study, we established a novel Ids-deficient mice and further assessed the enzyme's physiological role. Using DNA sequencing, we found a genomic modification of the Ids genome, which involved the deletion of a 138-bp fragment spanning from intron 2 to exon 3, along with the insertion of an adenine at the 5' end of exon 3 in the mutated allele. Consistent with previous data, our Ids-deficient mice showed an attenuated enzyme activity and an enhanced accumulation of glycosaminoglycans. Interestingly, we noticed a distinct enlargement of the calvarial bone in both neonatal and young adult mice. Our examination revealed that Ids deficiency led to an enhanced osteoblastogenesis in the parietal bone, a posterior part of the calvarial bone originating from the paraxial mesoderm and associated with an enhanced expression of osteoblastic makers, such as Col1a and Runx2. In sharp contrast, cell proliferation of the parietal bone in these mice appeared similar to that of wild-type controls. These results suggest that the deficiency of Ids could be involved in an augmented differentiation of calvarial bone, which is often noticed as an enlarged head circumference in MPS II-affected individuals.

Craniofacial development is a program exquisitely orchestrated by tissue contributions and regulation of genes expression. The basic helix-loop-helix (bHLH) transcription factor Twist1 expressed in the skeletal mesenchyme is a key regulator of craniofacial development playing an important role during osteoskeletogenesis. This study investigates the postnatal impact of Twist1 haploinsufficiency on the osteoskeletal ability and regeneration on two calvarial bones arising from tissues of different embryonic origin: the neural crest-derived frontal and the mesoderm-derived parietal bones. We show that Twist1 haplonsufficiency as well Twist1-sh-mediated silencing selectively enhanced osteogenic and tissue regeneration ability of mesoderm-derived bones. Transcriptomic profiling, gain-and loss-of-function experiments revealed that Twist1 haplonsufficiency triggers its selective activity on mesoderm-derived bone through a sharp downregulation of the bone-derived hormone Fgf23 that is upregulated exclusively in wild-type parietal bone.

As a culmination of efforts over the last years, our knowledge of the embryonic origins of the mammalian frontal and parietal cranial bones is unambiguous. Progenitor cells that subsequently give rise to frontal bone are of neural crest origin, while parietal bone progenitors arise from paraxial mesoderm. Given the unique qualities of neural crest cells and the clear delineation of the embryonic origins of the calvarial bones, we sought to determine whether mouse neural crest derived frontal bone differs in biology from mesoderm derived parietal bone.

Cell source is the key to decellularized matrix (DM) strategy. This study compared 3 cell types, osteocytes with/without dominant active Wnt/β-catenin signaling (daCO and WTO) and bone marrow stromal cells (BMSCs) for their DMs in bone repair. Decellularization removes all organelles and >95% DNA, and retained >74% collagen and >71% GAG, maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the BMSC-DM. DM produced higher cell survival rate (90%) and higher proliferative activity. In vitro, daCO-DM induces more and longer stress fibers in BMSCs, conducive to cell adhesion, spreading, and osteogenic differentiation. 8-wk after implantation of the critical-sized parietal bone defect model, daCO-DM formed tight structures, composed of a large number of densely-arranged type-I collagen under polarized light microscope, which is similar to and integrated with host bone. BV/TV (>54%) was 1.5, 2.9, and 3.5 times of WTO-DM, BMSC-DM, and none-DM groups, and N.Ob/T.Ar (3.2 × 102/mm2) was 1.7, 2.9, and 3.3 times. At 4-wk, daCO-DM induced osteoclastogenesis, 2.3 times higher than WTO-DM; but BMSC-DM or none-DM didn't. daCO-DM increased the expression of RANKL and MCSF, Vegfa and Angpt1, and Ngf in BMSCs, which contributes to osteoclastogenesis, angiogenesis, and neurogenesis, respectively. daCO-DM promoted H-type vessel formation and nerve markers β3-tubulin and NeuN expression. Conclusion: daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone defects. These features are expected to achieve the effect of autologous bone transplantation, suitable for transformation application.

Heterozygous mutations in MSX2 are responsible for an autosomal dominant form of parietal foramina (PFM). PFM are oval defects of the parietal bones that are also a characteristic feature of a contiguous gene-deletion syndrome caused by a proximal deletion in the short arm of chromosome 11 (Potocki-Shaffer syndrome). We have identified a human bacterial artificial chromosome (BAC) clone mapping to chromosome 11, containing a region homologous to the human homeobox gene MSX2. Further sequence analysis demonstrated that the human orthologue (ALX4) of the mouse Aristaless-like 4 gene (Alx4) is contained within this 11p clone. We used FISH to test for the presence-or for the heterozygous deletion-of this clone in two patients with the 11p11.2-deletion syndrome and showed that this clone is deleted in these patients. ALX4 and Alx4 were shown to be expressed in bone and to be absent from all other tissues tested. The involvement of Alx4 in murine skull development, its bone-specific expression pattern, the fact that Alx4 is a dosage-sensitive gene in mice, and the localization of a human genomic clone containing ALX4 to 11p11.2, with hemizygosity in patients with deletion of 11p11.2 who have biparietal foramina, support the contention that ALX4 is a candidate gene for the PFM in the 11p11.2-deletion syndrome.

Serum albumin-coated bone allografts (BoneAlbumin) have successfully supported bone regeneration in various experimental models by activating endogenous progenitors. However, the effect of tissue aging, linked to declining stem cell function, has yet to be explicitly examined within the context of BoneAlbumin's regenerative capacity. Stem cell function was tested with an in vitro attachment assay, which showed that albumin coating increases stem cell attachment on demineralized bone surfaces in an aging cell population. Bone regeneration was investigated in vivo by creating critical size bone defects on the parietal bones of aging female rats. Demineralized bone matrices with and without serum albumin coating were used to fill the defects. Bone regeneration was determined by measuring the density and the size of the remaining bone defect with computed tomography (CT). Microcomputed tomography (MicroCT) and mechanical testing were performed on the parietal bone explants. In vivo CT and ex vivo microCT measurements showed better regeneration with albumin-coated grafts. Additionally, the albumin-coated group showed a twofold increase in peak fracture force compared with uncoated allografts. In the present study, serum albumin-coated demineralized bone matrices successfully supported faster and functionally superior bone regeneration in aging rats. Because stem cell function, a key contributor of bone remodelling, decreases with age and serum albumin is an effective activator of endogenous progenitor cells, this method could be an effective and safe adjuvant in bone regeneration of aging adult and osteo-compromised populations.

The bone-anchored port (BAP) is an investigational implant, which is intended to be fixed on the temporal bone and provide vascular access. There are a number of implants taking advantage of the stability and available room in the temporal bone. These devices range from implantable hearing aids to percutaneous ports. During temporal bone surgery, injuring critical anatomical structures must be avoided. Several methods for computer-assisted temporal bone surgery are reported, which typically add an additional procedure for the patient. We propose a surgical guide in the form of a bone-thickness map displaying anatomical landmarks that can be used for planning of the surgery, and for the intra-operative decision of the implant's location. The retro-auricular region of the temporal and parietal bone was marked on cone-beam computed tomography scans and tridimensional surfaces displaying the bone thickness were created from this space. We compared this method using a thickness map (n = 10) with conventional surgery without assistance (n = 5) in isolated human anatomical whole head specimens. The use of the thickness map reduced the rate of Dura Mater exposition from 100% to 20% and suppressed sigmoid sinus exposures. The study shows that a bone-thickness map can be used as a low-complexity method to improve patient's safety during BAP surgery in the temporal bone.

Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest-derived frontal and paraxial mesodermal-derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest-derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal-derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest-derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF-2) treatment induces phosphorylation of GSK-3beta and increases the nuclear levels of beta-catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest-derived frontal bone.

Skeletal diseases and their surgical treatment induce severe pain. The innervation density of bone potentially explains the severe pain reported. Animal studies concluded that sensory myelinated A∂-fibers and unmyelinated C-fibers are mainly responsible for conducting bone pain, and that the innervation density of these nerve fibers was highest in periosteum. However, literature regarding sensory innervation of human bone is scarce. This observational study aimed to quantify sensory nerve fiber density in periosteum, cortical bone, and bone marrow of axial and appendicular human bones using immunohistochemistry and confocal microscopy. Multivariate Poisson regression analysis demonstrated that the total number of sensory and sympathetic nerve fibers was highest in periosteum, followed by bone marrow, and cortical bone for all bones studied. Bone from thoracic vertebral bodies contained most sensory nerve fibers, followed by the upper extremity, lower extremity, and parietal neurocranium. The number of nerve fibers declined with age and did not differ between male and female specimens. Sensory nerve fibers were organized as a branched network throughout the periosteum. The current results provide an explanation for the severe pain accompanying skeletal disease, fracture, or surgery. Further, the results could provide more insight into mechanisms that generate and maintain skeletal pain and might aid in developing new treatment strategies. PERSPECTIVE: This article presents the innervation of human bone and assesses the effect of age, gender, bone compartment and type of bone on innervation density. The presented data provide an explanation for the severity of bone pain arising from skeletal diseases and their surgical treatment.

Maintaining properties of human bone marrow-derived mesenchymal stem cells (BMSCs) in culture for regenerative applications remains a great challenge. An emerging approach of constructing a culture environment mimicking the bone marrow niche to regulate BMSC activities has been developed. In this study, we have demonstrated a systematic approach to identify soluble factors of interest extracted from human bone marrow and used them in BMSC culture for tissue regeneration. We have found that lipocalin-2 and prolactin are key factors in bone marrow, involved in regulating BMSC activities. Treating the cell with lipocalin-2 and prolactin delays cellular senescence of BMSCs and primes the cell for osteogenesis and chondrogenesis. We have also demonstrated that BMSCs pretreated with lipocalin-2 and prolactin can enhance the repair of calvarial defects in mice. Together, our study provides research evidence of using a viable approach to prime BMSC properties in vitro for improving cell-based tissue regeneration in vivo.

Fresh compact bone, the candidate graft material for bone regeneration, is usually grafted for horizontal bone augmentation. However, the dense calcified structure inhibits the release of growth factors and limits cellular and vascular perfusion. We aimed to create mechano-chemically altered dense skull bone by ultrasonic treatment, along with partial demineralization using commercially available acidic electrolyzed water (AEW). The parietal skull bone of an 11-month-old Wistar rat was exposed and continuously treated with a piezoelectric ultrasonic scaler tip for 1 min, using AEW (pH 2.3) or distilled water (DW, pH 5.6) as irrigants. Treated parietal bone was removed, cut into plates (5 × 5 × 1 mm3), grafted into the back subcutaneous tissues of syngeneic rats, and explanted at 1, 2, and 3 weeks. AEW bone showed an irregular surface, deep nano-microcracks, and decalcified areas. SEM-EDS revealed small amounts of residual calcium content in the AEW bone (0.03%) compared to the DW bone (0.86%). In the animal assay, the AEW bone induced bone at 2 weeks. Histomorphometric analysis showed that the area of new bone in the AEW bone at 2 and 3 weeks was significantly larger. This new combination technique of AEW-demineralization with ultrasonic treatment will improve the surface area and three-dimensional (3D) architecture of dense bone and accelerate new bone synthesis.

Hydrogels composed from biomolecules have gained great interests as biomaterials for tissue engineering. However, their poor mechanical properties limit their application potential. Here, we synthesized a series of tough composite hydrogels from poly (vinyl alcohol) (PVA) and pectin for bone tissue engineering. With a balance of scaffold stiffness and pore size, PVA-Pec-10 hydrogel enhanced adhesion and proliferation of osteoblasts. The hydrogel significantly promoted osteogenesis in vitro by improving the alkaline phosphates (ALP) activity and calcium biomineralization, as well as upregulating the expressions of osteoblastic genes. The composite hydrogel also accelerated the bone healing process in vivo after transplantation into the femoral defect. Additionally, our study demonstrated that pectin and its Ca2+ crosslinking network play a crucial role of inducing osteogenesis through regulating the Ca2+/CaMKII and BMP-SMAD1/5 signaling. The optimized structure composition and multifunctional properties make PVA-Pec hydrogel highly promising to serve as a candidate for bone tissue regeneration.

Enlarged fontanelles and smaller frontal bones result in a mechanically compromised skull. Both phenotypes could develop from defective migration and differentiation of osteoblasts in the skull bone primordia. The Wnt/Planar cell polarity (Wnt/PCP) signaling pathway regulates cell migration and movement in other tissues and led us to test the role of Prickle1, a core component of the Wnt/PCP pathway, in the skull. For these studies, we used the missense allele of Prickle1 named Prickle1Beetlejuice (Prickle1Bj). The Prickle1Bj/Bj mutants are microcephalic and develop enlarged fontanelles between insufficient frontal bones, while the parietal bones are normal. Prickle1Bj/Bj mutants have several other craniofacial defects including a midline cleft lip, incompletely penetrant cleft palate, and decreased proximal-distal growth of the head. We observed decreased Wnt/β-catenin and Hedgehog signaling in the frontal bone condensations of the Prickle1Bj/Bj mutants. Surprisingly, the smaller frontal bones do not result from defects in cell proliferation or death, but rather significantly delayed differentiation and decreased expression of migratory markers in the frontal bone osteoblast precursors. Our data suggests that Prickle1 protein function contributes to both the migration and differentiation of osteoblast precursors in the frontal bone.

To assess the effects of chronic alcoholism on the repair of bone defects associated with xenograft. Forty male rats were distributed in: control group (CG, n = 20) and experimental group (EG, n = 20), which received 25% ethanol ad libitum after a period of adaptation. After 90 days of liquid diet, the rats were submitted to 5.0-mm bilateral craniotomy on the parietal bones, subdividing into groups: CCG (control group that received only water with liquid diet and the defect was filled with blood clot), BCG (control group that received only water with liquid diet and the defect was filled with biomaterial), CEG (alcoholic group that received only ethanol solution 25% v/v with liquid diet and the defect was filled with blood clot), and BEG (alcoholic group that received only ethanol solution 25% v/v with liquid diet and the defect was filled with biomaterial). In the analysis of body mass, the drunk animals presented the lowest averages in relation to non-drunk animals during the experimental period. Histomorphologically all groups presented bone formation restricted to the defect margins at 60 days, with bone islets adjacent to the BCG biomaterial particles. CEG showed significant difference compared to BEG only at 40 days (17.42 ± 2.78 vs. 9.59 ± 4.59, respectively). In the birefringence analysis, in early periods all groups showed red-orange birefringence turning greenish-yellow at the end of the experiment. The results provided that, regardless of clinical condition, i.e., alcoholic or non-alcoholic, in the final period of the experiment, the process of bone defect recomposition was similar with the use of xenograft or only clot.

Growth, one of the most important traits monitored in domestic animals, is essentially associated with bone development. To date, no large-scale transcriptome studies investigating bone development in bighead carp have been reported. In this study, we applied Isoform-sequencing technology to uncover the entire transcriptomic landscape of the bighead carp (Hypophthalmichthys nobilis) in early growth stage, and obtained 63,873 non-redundant transcripts, 20,907 long non-coding RNAs, and 1,579 transcription factors. A total of 381 alternative splicing events were seen in the frontal and parietal bones with another 784 events simultaneously observed in the vertebral bones. Coupling this to RNA sequencing (RNA-seq) data, we identified 27 differentially expressed unigenes (DEGs) in the frontal and parietal bones and 45 DEGs in the vertebral bones in the fast-growing group of fish, when compared to the slow-growing group of fish. Finally, 15 key pathways and 20 key DEGs were identified and found to be involved in regulation of early growth such as energy metabolism, immune function, and cytoskeleton function and important cellular pathways such as the arginine and proline metabolic pathway (p4ha1), FoxO signaling pathway (sgk1), cell adhesion molecules (b2m, ptprc, and mhcII), and peroxisome proliferator-activated receptor signaling pathway (scd). We established a novel full-length transcriptome resource and combined it with RNA-seq to elucidate the mechanism of genetic regulation of differential growth in bighead carp. The key DEGs identified in this study could fuel further studies investigating associations between growth and bone development and serve as a source of potential candidate genes for marker-assisted breeding programs.

Biomaterial delivery of bioactive agents and manipulation of stem cell fate are an attractive approach to promote tissue regeneration. Here, smoothened agonist sterosome is developed using small-molecule activators [20S-hydroxycholesterol (OHC) and purmorphamine (PUR)] of the smoothened protein in the hedgehog pathway as carrier and cargo. Sterosome presents inherent osteoinductive property even without drug loading. Sterosome is covalently immobilized onto three-dimensional scaffolds via a bioinspired polydopamine intermediate to fabricate a hybrid scaffold for bone regeneration. Sterosome-immobilized hybrid scaffold not only provides a favorable substrate for cell adhesion and proliferation but also delivers bioactive agents in a sustained and spatially targeted manner. Furthermore, this scaffold significantly improves osteogenic differentiation of bone marrow stem cells through OHC/PUR-mediated synergistic activation of the hedgehog pathway and also enhances bone repair in a mouse calvarial defect model. This system serves as a versatile biomaterial platform for many applications, including therapeutic delivery and endogenous regenerative medicine.

Large-scale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. We present a comparative analysis of DNA preservation in these two substrates obtained from the same human skulls, across a range of different ages and preservation environments. Both substrates display significantly higher endogenous DNA content (average of 16.4% and 40.0% for teeth and petrous bones, respectively) than parietal skull bone (average of 2.2%). Despite sample-to-sample variation, petrous bone overall performs better than tooth cementum (p = 0.001). This difference, however, is driven largely by a cluster of viking skeletons from one particular locality, showing relatively poor molecular tooth preservation (<10% endogenous DNA). In the remaining skeletons there is no systematic difference between the two substrates. A crude preservation (good/bad) applied to each sample prior to DNA-extraction predicted the above/below 10% endogenous DNA threshold in 80% of the cases. Interestingly, we observe signficantly higher levels of cytosine to thymine deamination damage and lower proportions of mitochondrial/nuclear DNA in petrous bone compared to tooth cementum. Lastly, we show that petrous bones from ancient cremated individuals contain no measurable levels of authentic human DNA. Based on these findings we discuss the pros and cons of sampling the different elements.

Injectable hydrogels can fill irregular defects and promote in situ tissue regrowth and regeneration. The ability of directing stem cell differentiation in a three-dimensional microenvironment for bone regeneration remains a challenge. In this study, we successfully nanoengineer an interconnected microporous networked photocrosslinkable chitosan in situ-forming hydrogel by introducing two-dimensional nanoclay particles with intercalation chemistry. The presence of the nanosilicates increases the Young's modulus and stalls the degradation rate of the resulting hydrogels. We demonstrate that the reinforced hydrogels promote the proliferation as well as the attachment and induced the differentiation of encapsulated mesenchymal stem cells in vitro. Furthermore, we explore the effects of nanoengineered hydrogels in vivo with the critical-sized mouse calvarial defect model. Our results confirm that chitosan-montmorillonite hydrogels are able to recruit native cells and promote calvarial healing without delivery of additional therapeutic agents or stem cells, indicating their tissue engineering potential.

The erbium-doped yttrium aluminium garnet (Er:YAG) laser has emerged as a possible alternative to conventional methods of bone ablation because of its wavelength of 2.94 μm, which coincides with the absorption peak of water. Over the last decades in several experimental and clinical studies, the widespread initial assumption that light amplification for stimulated emission of radiation (laser) osteotomy inevitably provokes profound tissue damage and delayed wound healing has been refuted. In addition, the supposed disadvantage of prolonged osteotomy times could be overcome by modern short-pulsed Er:YAG laser systems. Currently, the limiting factors for a routine application of lasers for bone ablation are mainly technical drawbacks such as missing depth control and a difficult and safe guidance of the laser beam. This article gives a short overview of the development process and current possibilities of noncontact Er:YAG laser osteotomy in oral and implant surgery.