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Stem cell therapy could potentially replace damaged heart muscle for regenerative cardiology. We hypothesized that immature cardiovascular progenitors (CVPs) when transplanted into infarcted hearts would electrically synchronized and mature in vivo thus avoiding the occurrence of VTs. In this study, we reported the derivation and applications of hESC-derived CVPs using highly reproducible (R 2 > 0.95) laminin-221 differentiation protocol. The infarction model was created on 3 months old Sus scrofa pigs where we permanently ligated the coronary arteries and intramyocardially transplanted 200 million CVPs. A total of 3 sham, 10 medium control and 10 CVP transplanted pigs were monitored at 4- and 12-weeks post-transplantation. We demonstrated using 10X spatial transcriptomic analysis at 1-week following transplantation the engraftment of the human CVPs into the pig heart. The cells remained viable and proliferated to formed human graft in the infarcted region at 4- and 12-weeks as indicated by IVIS imaging and histology staining. Heart function was analyzed by magnetic resonance imaging (MRI) and revealed overall improvement in left ventricular ejection fraction by 21.35 ± 3.3 %, which was accompanied by significant improvements in ventricular wall thickness and wall motion, as well as a reduction in infarction size after CVP transplantation as compared to medium control pigs (P < 0.05). Electrophysiology analyses revealed electrical propagation between transplanted cells and host tissue CMs. Temporary episodes of VT over a period of 25 days were developed in 4 out of 10 CVP transplanted pigs and 1 pig had persistent VT, while the rest (n = 5) remained in normal sinus rhythm. All ten pigs survived the experiment without any VT-related death. Finally, computerized tomography (CT) scans revealed no tumor formation after 12-weeks transplantation indicating the long-term safety of the treatment. In conclusion, we reported the generation of an effective and potent CVPs and a significant reduction (50 %) in graft induced VT as compared to studies transplanted with contracting cardiomyocytes. This method may pave the way for stem cell therapy of myocardial infarction in humans.

BackgroundIschemic heart disease is a huge global burden where patients often have irreversibly damaged heart muscle. State-of-the-art technology using stem cell-derived products for cellular therapy could potentially replace damaged heart muscle for regenerative cardiology.Methods and ResultsPluripotent human embryonic stem cells (hESCs) were differentiated on a laminin LN521+221 matrix to cardiovascular progenitors (CVPs). Global transcriptome analyses at multiple time points by single-cell RNA-sequencing demonstrated high reproducibility (R2 > 0.95) between two hESCs lines. We identified several CVP signature genes as quality batch control parameters which are highly specific to our CVPs as compared to canonical cardiac progenitor genes. A total of 200 million CVPs were injected into the infarcted region caused by permanent ligation of the coronary arteries of 10 immunosuppressed pigs and maintained for 4- and 12-weeks post transplantation. The transplanted cells engrafted and proliferated in the infarcted area as indicated by IVIS imaging, histology staining and spatial transcriptomic analysis. Spatial transcriptomic analysis at 1 week following transplantation showed that the infarcted region expressed human genes in the same area as immunohistology sections. Heart function was analyzed by magnetic resonance imaging (MRI) and computerized tomography (CT). Functional studies revealed overall improvement in left ventricular ejection fraction by 21.35 ± 3.3 %, which was accompanied by significant improvements in ventricular wall thickness and wall motion, as well as a reduction in infarction size after CVP transplantation as compared to medium control pigs (P < 0.05). Immunohistology analysis revealed maturation of the CVPs to cardiomyocytes (CMs) where the human grafts aligned with host tissue forming end-to-end connections typical for heart muscle. Electrophysiology analyses revealed electric continuity between injected and host tissue CMs. Episodes of ventricular tachyarrhythmia (VT) over a period of 25 days developed in four pigs, one pig had persistent VT, while the rest remained in normal sinus rhythm. All ten pigs survived the experiment without any VT-related death.ConclusionsWe report a highly reproducible, chemically defined and fully humanized differentiation method of hESCs for the generation of potent CVPs. This method may pave the way for lasting stem cell therapy of myocardial infarction (MI) in humans.Clinical PerspectiveWhat is New?We present a highly reproducible, chemically defined and fully humanized laminin-based differentiation method for generation of large amounts of cardiovascular progenitors (CVP); 20 million cells in a 10 cm2 culture dish which were used for a preclinical study in pigs.Transplantation of the CVPs into the myocardial infarcted pig hearts yields maturation of the progenitor cells to cardiomyocytes (CMs) and improved cardiac function (21.35 ± 3.3 % LVEF improvement) using only 200 million CVPs.Temporary episodes of ventricular arrhythmia (50%) were observed after CVP transplantation. No fatal ventricular arrhythmia occurred.What are the clinical implications?Our laminin-based approach generated potent CVPs in vivo and largely restored function of the damaged heart.Cardiovascular progenitors may provide a new and safe therapeutic strategy for myocardial infarction.The results may have a significant impact on regenerative cardiology.

Regeneration of human heart muscle after injury is extremely limited and an unmet clinical need. Despite extensive research, there are no methods for the reproducible generation of clinical quality stem-cell-derived cardiovascular progenitors (CVPs). Here, we identified laminin-221 (LN-221) as the main cardiac laminin, which was produced here as human recombinant protein, and showed that LN-221 promotes differentiation of pluripotent hESCs towards cardiomyocyte lineage and downregulates genes associated with pluripotency and teratoma development. We developed a chemically defined, xeno-free laminin-based cardiomyocyte differentiation protocol to reproducibly generate CVPs that form human muscle in vivo. We assessed the reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from two different hESC lines. Single-cell RNA sequencing of CVPs derived from two hESC lines further showed high reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical quality cells for use in regenerative cardiology.

The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system. Through our characterization efforts of the human skin basement membrane and murine feeder layer 3T3-J2, we identified two biologically relevant recombinant laminins—LN-511 and LN-421- as potential candidates to replace the murine feeder. Herein, we report a completely xeno-free and defined culture system utilizing these laminins which enables robust expansion of adult human skin keratinocytes. We demonstrate that our laminin system is comparable to the 3T3-J2 co-culture system in terms of basal markers’ profile, colony-forming efficiency and the ability to form normal stratified epidermal structure in both in vitro and in vivo models. These results show that the proposed system may not only provide safer keratinocyte use in the clinics, but also facilitate the broader use of other cultured human epithelial cells in regenerative medicine., In vitro expansion of human epidermal keratinocytes to resurface severe wound defects still relies on a human/mouse xenograft culture system. Here the authors develop a fully human, xeno-free culture system using skin-associated laminins, normally present in vivo, to replace mouse feeder cells.

The efficacy of islet transplantation for diabetes treatment suffers from lack of cadaver-derived islets, islet necrosis and long transfer times prior to transplantation. Here, we developed a method for culturing mouse and human islets in vitro on α5-laminins, which are natural components of islet basement membranes. Adhering islets spread to form layers of 1–3 cells in thickness and remained normoxic and functional for at least 7 days in culture. In contrast, spherical islets kept in suspension developed hypoxia and central necrosis within 16 h. Transplantation of 110–150 mouse islets cultured on α5-laminin-coated polydimethylsiloxane membranes for 3–7 days normalized blood glucose already within 3 days in mice with streptozotocin-induced diabetes. RNA-sequencing of isolated and cultured mouse islets provided further evidence for the adhesion and spreading achieved with α5-laminin. Our results suggest that use of such in vitro expanded islets may significantly enhance the efficacy of islet transplantation treatment for diabetes.

The study evaluates the osteogenic properties and biocompatibility of growth factor-rich demineralized bone matrix (GDBM) by comparing with cancellous mineralized bone matrix (CMBM) and anorganic bovine bone matrix (ABBM).Thirty-six Sprague-Dawley rats were used (n = 6/group/time point). To assess biocompatibility and osteoinductivity, the respective bone matrices were randomly placed in subcutaneous pouches for 7 and 28 days and evaluated by histology and osteopontin expression. Osteoconductivity was assessed by randomly implanting respective bone matrices in osteotomies on femurs for 14 and 28 days and evaluated by microcomputed tomography and histology.Neither acute inflammation nor mineralized tissue was noted in any of the subcutaneous specimens, whereas expression of osteopontin was more prominent in the GDBM group. Among the femoral specimens, the greatest relative bone volume (bone volume [BV] divided by trabecular volume [TV]) and trabecular thickness was noted in the ABBM group at both time points, whereas less BV/TV was noted in GDBM group at day 14. Residual matrix particles were noted in all examined groups at both time points, without significant differences regarding defect fill between groups. The GDBM group presented similar levels of newly formed bone compartment and marrow space to those of the ABBM group.GDBM demonstrated acceptable biocompatibility and osteogenic potential comparable to ABBM in vivo. Further investigations in a more clinically relevant model are warranted.

Advanced glycation end products (AGE) have been found in inflamed gingival tissue and have been shown to interfere with the integrity of extracellular matrix and cell-matrix interactions. This study aims to investigate the modulatory effect of aminoguanidine (AG), an AGE inhibitor, in various stages of experimental periodontitis.Thirty-six Sprague-Dawley rats were used. AG or normal saline (NS) was systemically administered in the induction, progression, and recovery phases of ligature-induced periodontitis. Dynamic changes of the periodontium were evaluated by microcomputed tomography, histology, and immunohistochemistry of the receptor for AGE (RAGE). Molecular mechanisms were evaluated by myeloperoxidase activity, gene expression of RAGE, and markers associated with tissue repair and homeostasis, including vascular endothelial growth factor (VEGF), type I collagen, fibronectin, and periostin.AG appeared to inhibit the degradation of the collagen matrix in the induction phase but promoted collagen reorganization in the progression and recovery phases of experimental periodontitis. In the induction sites, periodontal bone loss was significantly reduced (P0.05), with significantly reduced RAGE (P0.05) and significantly elevated fibronectin and periostin levels (P0.01). No significant alterations in the levels of myeloperoxidase, VEGF, and collagen were noted. In the progression and recovery sites, similar trends were observed, with insignificant differences relative to NS-treated animals.AG reduced periodontal bone loss during the induction of experimental periodontitis, and the effects appeared to be insignificant in the progression and recovery phases. This modulation was related to the inhibition of the AGE-RAGE axis to resume cell-matrix interactions and maintain tissue integrity.

The emphasis on periodontal regeneration has been shifted towards the harmonization of bioactive molecules and physiological phases during regeneration. This study investigated whether the combination and sequential-release of platelet-derived growth factor (PDGF, mitogen) and simvastatin (differentiation factor) facilitated periodontal regeneration. PDGF and simvastatin were encapsulated in double-walled poly-( d,l-lactide) and poly-(d,l-lactide-co-glycolide) (PDLLA-PLGA) microspheres using the co-axial electrohydrodynamic atomization technique. Critical-sized periodontal defects on rat maxillae were filled with microspheres encapsulating BSA-in-core-shell (BB), PDGF-in-shell (XP), simvastatin-in-core and BSA-in-shell (SB), simvastatin-in-core and PDGF-in-shell, or unfilled with microspheres (XX), and examined at 14 and 28 days post-operatively. The resultant microspheres were around 15 μm diameter with distinct core-shell structure, and the fast-release of PDGF followed by slow-release of simvastatin was noted in the SP group. The SP group demonstrated significantly greater bone volume fraction and decreased trabecular separation compared to the XX group at day 14, and milder inflammatory cells infiltration and elevated tartrate-resistant acid phosphatase level were noted at day 28. Fibers were also well-aligned and obliquely inserted onto the root surface similar to native periodontal ligament with signs of cementogenesis in the SP group. In conclusion, the combination and sequential-release of PDGF-simvastatin accelerates the regeneration of the periodontal apparatus.

Because a single exposure to light-emitting diode (LED) irradiation at 660 nm only demonstrated a 3-day biostimulatory effect in recovering periodontal bone level (PBL), this study sought to evaluate whether the periodontal effect could be extended through the use of multiple LED irradiations. Experimental periodontitis was developed unilaterally in 48 Sprague–Dawley rats after the placement of a silk ligature plus Porphyromonas gingivalis lipopolysaccharide injections. The animals were divided into four groups (no irradiation, a single irradiation, or two or three irradiations per week) and exposed to LED light irradiation at a wavelength of 660 ± 25 nm and energy density of 10 J/cm2 after debridement and detoxification. The animals were euthanized after 7 or 14 days, and the effect of irradiation was evaluated using micro-computed tomography and histology. By day 7, PBL was significantly reduced (p < 0.05), with significantly reduced inflammation (p < 0.05) and gingival hyperplasia (p < 0.001), in the animals receiving three irradiations per week. At day 14, the reduction in gingival hyperplasia was still significant (p < 0.05), and collagen matrix deposition and realignment appeared to be accelerated in the animals receiving three irradiations per week, despite a lack of significant difference in PBL. The treatment regimen receiving three LED light irradiations per week apparently extended the effects in reducing PBL and inflammation to 7 days. The inclusion of additional inflammation control measures or the addition of bioactive signals to mediate the repairing process is necessary to maintain long-term periodontal stability.

Proper coordination of local signal to harmonize mitogenesis and osteogenic differentiation is one of the prerequisites to optimize dentoalveolar regeneration. In the study, we purpose to fabricate controlled-release microspheres encapsulating platelet-derived growth factor (PDGF) and simvastatin by coaxial electrohydrodynamic atomization. The microspheres demonstrated a distinct core and shell structure encapsulating PDGF and simvastatin respectively, and the encapsulation efficiency was 82.45–92.16% in-core and 51.37–71.34% in-shell. Sequential release of PDGF and simvastatin was seen in simvastatin-in-core and PDGF-in-shell (SP) design, and simultaneous release was achieved in PDGF-in-core and simvastatin-in-shell (PS) design. All microspheres demonstrated acceptable biocompatibility in vivo, with increased proliferation, reduced apoptosis, and reduced inflammation while PDGF or simvastatin was encapsulated. The PS design significantly reduced apoptosis than control, whereby significant and persistent enhanced proliferation was noted in SP group. The thickness of fibrotic capsules surrounding microspheres significantly reduced in both SP and PS group at day 14. The finding demonstrates that synergism of PDGF and simvastatin favored biocompatibility. Further investigations will aim on confirming the regenerative effect of SP and PS microspheres in a more clinically relevant model. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:2970–2978, 2012.

Obesity is a global epidemic that contributes to the increasing medical burdens related to type 2 diabetes, cardiovascular disease and cancer. A better understanding of the mechanisms regulating adipose tissue expansion could lead to therapeutics that eliminate or reduce obesity-associated morbidity and mortality. The extracellular matrix (ECM) has been shown to regulate the development and function of numerous tissues and organs. However, there is little understanding of its function in adipose tissue. In this manuscript we describe the role of laminin α4, a specialized ECM protein surrounding adipocytes, on weight gain and adipose tissue function. Adipose tissue accumulation, lipogenesis, and structure were examined in mice with a null mutation of the laminin α4 gene (Lama4−/ −) and compared to wild-type (Lama4+/+) control animals. Lama4−/ − mice exhibited reduced weight gain in response to both age and high fat diet. Interestingly, the mice had decreased adipose tissue mass and altered lipogenesis in a depot-specific manner. In particular, epididymal adipose tissue mass was specifically decreased in knock-out mice, and there was also a defect in lipogenesis in this depot as well. In contrast, no such differences were observed in subcutaneous adipose tissue at 14 weeks. The results suggest that laminin α4 influences adipose tissue structure and function in a depot-specific manner. Alterations in laminin composition offers insight into the roll the ECM potentially plays in modulating cellular behavior in adipose tissue expansion.

Advanced glycation end products (AGEs) are involved in the inflammatory process and are considered to be etiologic factors of diabetic periodontitis. The purpose of this study is to investigate the capability of N-phenacylthiazolium bromide (PTB), a glycated cross-link breaker, in the modulation of periodontitis in various disease phases.Mitogenesis and cytotoxicity of human periodontal ligament cells (hPDLCs) undergoing PTB treatment were evaluated in vitro. In vivo biomodulation was investigated by systemically administering PTB in the induction, progression, and recovery phases of ligature-induced periodontitis in rats, with the results evaluated by microcomputed tomography, histology, immunohistochemistry of the AGE and AGE receptor (RAGE), and gene expression of tumor necrosis factor-α (TNF-α), RAGE, periostin, fibronectin, and type I collagen.Significantly promoted mitogenesis and reduced cytotoxicity of hPDLCs were noted with 0.05 to 0.1 mM PTB treatment at 24 hours. Systemic PTB administration significantly reduced periodontal bone loss, AGE deposition, and expressions of TNF-α and RAGE but elevated the periostin level in all three phases of periodontitis.PTB inhibits the induction and progression of periodontitis and facilitates its recovery via improving cellular viability and inhibiting the AGE-RAGE axis.

Objectives: Timely augmentation of the physiological events of dentoalveolar repair is a prerequisite for the optimization of the outcome of regeneration. This study aimed to develop a treatment strategy to promote dentoalveolar regeneration by the combined delivery of the early mitogenic factor platelet-derived growth factor (PDGF) and the late osteogenic differentiation factor simvastatin. Materials and Methods: By using the coaxial electrohydrodynamic atomization technique, PDGF and simvastatin were encapsulated in a double-walled poly(d,l-lactide) and poly(d,l-lactide-co-glycolide) (PDLLA–PLGA) microspheres in five different modes: microspheres encapsulating bovine serum albumin (BB), PDGF alone (XP), simvastatin alone (SB), PDGF-in-core and simvastatin-in-shell (PS), and simvastatin-in-core and PDGF-in-shell (SP). The microspheres were characterized using scanning electronic microscopy, and the in vitro release profile was evaluated. Microspheres were delivered to fill large osteotomy sites on rat maxillae for 14 and 28 days, and the outcome of regeneration was evaluated by microcomputed tomography and histological assessments. Results: Uniform 20-μm controlled release microspheres were successfully fabricated. Parallel PDGF–simvastatin release was noted in the PS group, and the fast release of PDGF followed by the slow release of simvastatin was noted in the SP group. The promotion of osteogenesis was observed in XP, PS, and SP groups at day 14, whereas the SP group demonstrated the greatest bone fill, trabecular numbers, and thickest trabeculae. Bone bridging was evident in the PS and SP group, with significantly increased osteoblasts in the SP group, and osteoclastic cell recruitment was promoted in all bioactive molecule-treated groups. At day 28, osteogenesis was promoted in all bioactive molecule-treated groups. Initial corticalization was noted in the XP, PS, and SP groups. Osteoblasts appeared to be decreased in all groups, and significantly, a greater osteoclastic cell recruitment was noted in the SB and SP groups. Conclusions: Both PDGF and simvastatin facilitate dentoalveolar regeneration, and sequential PDGF–simvastatin release (SP group) further accelerated the regeneration process through the enhancement of osteoblastogenesis and the promotion of bone maturation.

The success of periodontal regeneration depends on the coordination of early cell proliferation and late cell differentiation. The aim of this study was to investigate whether the proliferation or differentiation stage predominantly promotes the initiation of periodontal regeneration. Critical-sized periodontal defects were surgically created on rat maxillae and filled with poly-(D,L-lactide-co-glycolide)-poly-d,l-lactide hybrid microspheres encapsulating platelet-derived growth factor (PDGF, a promoter of mitogenesis), simvastatin (a promoter of osteogenic differentiation), or bovine serum albumin (a control). The encapsulation efficiency and in vitro release profiles of the microspheres were determined by high-performance liquid chromatography and enzyme-linked immunosorbent assay. The maxillae were harvested after 10 or 14 days and assessed by micro-computed tomography, histology, and immunohistochemistry for regeneration efficacy and cell viability. The rapid release of PDGF was observed within the first week, whereas a slow release profile was noted for simvastatin. The PDGF-treated specimens demonstrated a significantly higher bone volume fraction compared with bovine serum albumin- (p0.05) or simvastatin-treated (p0.05) specimens at day 14. Histologically, active bone formation originating from the defect borders was noted in both the PDGF- and the simvastatin-treated specimens, and functionally aligned periodontal ligament fiber insertion was only observed in the PDGF-treated specimens. The significant promotion of mitogenesis by PDGF treatment was also noted at day 14 (p0.05). In conclusion, increased mitogenesis or osteogenic differentiation may stimulate osteogenesis, and the upregulation of mitogenesis by PDGF appears to play a role in the initiation of periodontal regeneration.

Background and Objective Hyperglycemia and advanced glycation end-products (AGEs) have been hypothesized as the etiologic factors of diabetic periodontitis. The aim of this study was to clarify in greater detail the patterns of AGE-mediated periodontal inflammation under various physiological conditions. Material and Methods The deposition of AGEs and expression of the receptor for AGEs (RAGE) were identified by immunohistochemistry in Sprague–Dawley rats with experimentally induced periodontitis or diabetes. Human periodontal ligament cells (PDLCs) and mesenchymal stem cells (MSCs) were cultured under simulated conditions of hyperglycemia, Porphyromonas gingivalis lipopolysaccharide (LPS) stimulation and matrix glycation. Cell viability and expression of toll-like receptors (TLRs), Rage, an inflammatory signaling initiator (nuclear factor kappa light chain enhancer of activator β cells), an oxidative stressor (heme oxygenase-1) and collagen synthesis (type I and type IV) genes were evaluated. Results The deposition of AGEs and the expression of Rage were evident in the inflamed periodontal tissues in all rats and appeared to be enhanced in rats with diabetes. Matrix glycation augmented cytotoxicity, up-regulated RAGE and TLRs in both PDLCs and MSCs, and significantly activated downstream inflammatory signaling in MSCs. Oxidative stress was significantly increased under matrix glycation in both PDLCs and MSCs and was significantly increased at a high-glucose concentration in MSCs. A consistent decrease in expression of type I and type IV collagens was observed in MSCs, but a delayed reduction was noted in PDLCs. Conclusions Matrix glycation modulated cell behavior to induce inflammation equivalent to that produced by incubation with P. gingivalis LPS. Periodontal inflammation also led to matrix glycation, thus demonstrating a definite interaction between diabetes and periodontitis.

Progression of diabetes-associated periodontal destruction and the roles of advanced glycation end products (AGEs) are investigated.Diabetes was induced by streptozocotin injection, and periodontitis was induced via silk ligature placement with Porphyromonas gingivalis lipopolysaccharide injection in 64 Sprague-Dawley rats for 7 to 21 days. The quality of alveolar bone and attachment loss (AL) were measured by microcomputed tomography and histology. Destruction profiles were evaluated by histology, histochemistry, immunohistochemistry, and quantitative assessments of inflammatory cells, expression of receptors for AGEs (RAGE), tartrate-resistant acid phosphatase, and proliferating cell nuclear antigen.Without periodontitis induction, there was no obvious morphologic change in the periodontium, although slight elevations of AGEs and RAGE levels were noted in animals with diabetes. In the group with experimental periodontitis, significant periodontal bone loss was noted in animals both with and without diabetes from day 7, with more progressive bone loss in animals with diabetes during days 14 to 21. Histologically, the disruption of attachment and inflammation were observed from day 7, but subsequently subsided in animals without diabetes. A stronger and more prolonged response with significant AL was observed in animals with diabetes. Stronger inflammation, attenuated and persistent resorptive activity, and weaker proliferating potential were demonstrated by animals with diabetes. AGE deposition and RAGE expression were noted in animals without diabetes but with periodontitis, although levels were considerably elevated in the later stages in animals with diabetes.Diabetes augments periodontal destruction by reducing the proliferating capability and activating resorptive activities. Presence of the AGE-RAGE axis without diabetes implies that it is involved in the regulation of inflammation.

Diabetes is known to impair wound healing and deteriorate the periodontal condition. There is limited information about the patterns and events associated with periodontal wound repair. In this study, we evaluate the dynamics of periodontal wound repair using micro-computed tomography (microCT) and immunohistochemistry.Thirty-six male rats were used, and diabetes was induced by streptozotocin. The maxillary first molars were extracted, and a tooth-associated osseous defect was created in the extraction area. Animals were sacrificed after 7, 14, and 21 days. Volumetry and distribution of bone trabeculae were evaluated by microCT imaging. The patterns of healing and collagen alignment were evaluated by histology. Advanced glycation end-product (AGE) deposition and expression of the receptor for AGEs (RAGE), tartrate-resistant acid phosphatase, and proliferating cell nuclear antigen were evaluated by histochemical and immunohistochemical staining.Diabetic animals demonstrated a significantly reduced bone volume and trabecular number as well as thinner trabeculae and more trabecular separation in osseous defects. The early stage was characterized by significantly reduced cellular proliferation and prolonged active inflammation without evident bone resorption, whereas delayed recovery of collagen realignment, matrix deposition, and bone turnover was noted in later stages. Although AGEs and RAGE were present during healing in diabetes and controls, a stronger and more persistent level of expression was observed in the group with diabetesDiabetes significantly delayed osseous defect healing by augmenting inflammation, impairing proliferation, and delaying bone resorption. The AGE-RAGE axis can be activated under metabolic disturbance and inflammation.