Buffering Mitigates Chondrocyte Oxidative Stress, Metabolic Dysfunction, and Death Induced by Normal Saline: Formulation of a Novel Arthroscopic Irrigant.

For decades, surgeons have utilized 0.9% normal saline (NS) for joint irrigation to improve visualization during arthroscopic procedures. This continues despite mounting evidence that NS exposure impairs chondrocyte metabolism and compromises articular cartilage function. We hypothesized that chondrocyte oxidative stress induced by low pH is the dominant factor driving NS toxicity, and that buffering NS to increase its pH would mitigate these effects. Effects on chondrocyte viability, reactive oxygen species (ROS) production, and overall metabolic function were assessed. Even brief exposure to NS caused cell death, ROS overproduction, and disruption of glycolysis, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways. NS also stimulated ROS overproduction in synovial cells that could adversely alter the synovial function and subsequently the entire joint health. Buffering NS with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) significantly increased chondrocyte viability, reduced ROS production, and returned metabolite levels to near control levels while also reducing ROS production in synovial cells. These results confirm that chondrocytes and synoviocytes are vulnerable to insult from the acidic pH of NS and demonstrate that adding a buffering agent to NS averts many of its most harmful effects.

Treatment acceptance in adult patients with anterior open bite: A National Dental Practice-Based Research Network study.

INTRODUCTION: Orthodontists have many techniques to treat anterior open bites and must involve patients in making treatment decisions. This study aimed to investigate orthodontic treatment plan acceptance by United States adults with anterior open bites and to identify associations between treatment acceptance and patient demographics and dentofacial characteristics. METHODS: A prospective, observational cohort study enrolled the patients of 91 orthodontic providers. A total of 345 adults were included in the sample. Provider personal and practice demographics were captured. Patient demographics, treatment goals, dentofacial characteristics, and reasons for not accepting the recommended plans were recorded. Adjusted regression models were used to identify associations between patient characteristics and the likelihood of accepting the most highly recommended plan. RESULTS: Approximately 78% of patients accepted the most highly recommended treatment plan. 60% of the patients who were recommended surgical plans accepted them. Patients with a history of orthodontics and a concave profile were more likely to accept the most highly recommended plan. Insurance coverage for orthognathic surgery was associated with a higher acceptance rate for surgery. Severe crowding was associated with a lower acceptance of surgery. The most common reasons for declining the recommended plan were not wanting jaw surgery and considering the treatment to be too invasive, risky, and/or costly. CONCLUSIONS: Patient acceptance is less common at higher levels of invasiveness of treatment. Prior orthodontic treatment, concave profile, and insurance coverage for surgery were associated with accepting treatment. Most patients accepted a surgical plan when it was the most recommended option.

Exosome-Based Cell Homing and Angiogenic Differentiation for Dental Pulp Regeneration.

Exosomes have attracted attention due to their ability to promote intercellular communication leading to enhanced cell recruitment, lineage-specific differentiation, and tissue regeneration. The object of this study was to determine the effect of exosomes on cell homing and angiogenic differentiation for pulp regeneration. Exosomes (DPSC-Exos) were isolated from rabbit dental pulp stem cells cultured under a growth (Exo-G) or angiogenic differentiation (Exo-A) condition. The characterization of exosomes was confirmed by nanoparticle tracking analysis and an antibody array. DPSC-Exos significantly promoted cell proliferation and migration when treated with 5 × 108/mL exosomes. In gene expression analysis, DPSC-Exos enhanced the expression of angiogenic markers including vascular endothelial growth factor A (VEGFA), Fms-related tyrosine kinase 1 (FLT1), and platelet and endothelial cell adhesion molecule 1 (PECAM1). Moreover, we identified key exosomal microRNAs in Exo-A for cell homing and angiogenesis. In conclusion, the exosome-based cell homing and angiogenic differentiation strategy has significant therapeutic potential for pulp regeneration.

The National Dental Practice-Based Research Network Adult Anterior Open Bite Study: Patient satisfaction with treatment.

INTRODUCTION: This article evaluates and reports on the satisfaction of adult patients across the United States who received orthodontic treatment for anterior open bite malocclusion. The factors that influence satisfaction are also described. METHODS: Practitioners were recruited from the National Dental Practice-Based Research Network. On joining the Network, practitioner demographics and information on their practices were acquired. Practitioners enrolled their adult patients in active treatment for anterior open bite. Patient demographics, patient dentofacial characteristics, and details regarding previous and current treatment were collected through questionnaires at enrollment (T1). Pretreatment lateral cephalograms and intraoral frontal photographs were submitted. Treatment performed, and details related to treatment outcome were recorded through questionnaires at the end of active treatment (T2). Posttreatment lateral cephalograms and intraoral frontal photographs were submitted. Patient satisfaction at T2 was assessed using a 5-point Likert-type scale and open-ended responses. Predictive univariate models were developed to evaluate the factors that affect patient satisfaction. Open-ended responses were reviewed for general trends. RESULTS: T2 data were received for 260 patients, and 248 of these patients completed and returned the patient satisfaction questionnaires. High levels of satisfaction were found in this sample of adult patients receiving treatment for anterior open bite malocclusion. Specifically, 96% of the sample reported being very or somewhat satisfied. Only 10 patients (4%) were not satisfied with the treatment provided or an element of the final result. Successful open bite closure, treatment modality, and certain patient characteristics may affect patient satisfaction. However, there was insufficient power to demonstrate statistical significance because of the very low number of dissatisfied patients. Open-ended responses directly associated with patient satisfaction were received from 23 patients (9%). They relayed positive, neutral, and negative feelings about the treatment received and final results. Additional responses regarding the orthodontic treatment in general, but not specifically linked to patient satisfaction, were received from 119 patients (48%). These comments depict an overwhelmingly positive experience. CONCLUSIONS: Adult patients who received orthodontic treatment for anterior open bite malocclusion were generally satisfied with the treatment provided, as well as the final esthetic and functional results.

The National Dental Practice-Based Research Network Adult Anterior Open Bite Study: Treatment success.

INTRODUCTION: Anterior open bite (AOB) continues to be a challenging malocclusion for orthodontists to treat and retain long-term. There is no consensus on which treatment modality is most successful. This study reports on the overall success rate of AOB orthodontic treatment in the adult population across the United States, as well as 4 major treatment modalities and other factors that may influence treatment success. METHODS: Practitioners and their adult patients with AOB were recruited through the National Dental Practice-Based Research Network. Patient dentofacial and demographic characteristics, practitioner demographic and practice characteristics, and factors relating to orthodontic treatment were reported. Treatment success was determined from posttreatment (T2) lateral cephalometric films and intraoral frontal photographs. Treatment was categorized into 4 main groups: aligners, fixed appliances, temporary anchorage devices (TADs), and orthognathic surgery. Extractions were also evaluated. Bivariate and multivariable models were used to investigate the association between treatment success and treatment modality, pretreatment (T1) dentofacial characteristics, patient and practitioner demographics, and practice characteristics, adjusting for clustering of patients within practice. RESULTS: A total of 254 patients, enrolled by 84 practitioners, contributed to T2 data for this study. There were 29 patients in the aligner group, 152 in fixed appliances, 20 in TADs, and 53 in surgery. A total of 49 patients underwent extractions of teeth other than third molars. Ninety-three percent finished treatment with a positive overbite on the T2 lateral cephalogram, and 84% finished with a positive vertical overlap of all incisors. The small number of aligners and TAD patients limited the ability to compare success rates in these groups. Patients treated with orthognathic surgery had a higher rate of success compared with those treated with fixed appliances only. Treatment success was also associated with academic practice setting, T1 mandibular plane angle ≤30°, no to mild T1 crowding, and treatment duration <30 months. CONCLUSIONS: The overall success of orthodontic treatment in adult patients with AOB who participated in this study was very high. Orthognathic surgery was the only treatment modality that exhibited a statistically higher odds of successful outcomes. Some T1 dentofacial characteristics and treatment factors were associated with the successful closure of AOB.

No Clear Evidence for the Effect of Low-Level Laser Treatment on Orthodontically Induced Inflammatory Root Resorption: More Human Clinical Studies Are Needed.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: Influence of low-level laser therapy on orthodontically induced inflammatory root resorption. A systematic review. Michelogiannakis D, l-Shammery D, Akram Z, Rossouw PE, Javed F, Romanos GE. Arch Oral Biol 2019;100:1-13. SOURCE OF FUNDING: The authors reported that this article did not receive any specific grants from funding agencies in the public, commercial, or nonprofit sectors. TYPE OF STUDY/DESIGN: Systematic review.

Characteristics of an excellent orthodontic residency program.

INTRODUCTION: Although unquantifiable features, such as faculty passion and dedication to teaching, play a vital role in defining the quality of residency education, determinable features that are fundamental to the definition of a "top tier" orthodontic residency program also exist. The objective of this study was to identify those features. METHODS: A survey with 32 items was developed and validated to assess the features of an excellent orthodontic program based on the following 3 major domains: faculty, education, and resident/graduate student/alumni. The survey was sent to 62 orthodontic residency programs in the United States. RESULTS: Thirty-nine programs (63%) completed the survey. Recurring attributes that were identified in what constitutes an excellent program included the following: an adequate number of full-time clinical orthodontic faculty, with each member providing 1 day per week clinic coverage. The average of all respondents was 4, and the range was 1-6; a healthy mix of part-time faculty members with ≥1 full-time faculty member who monitors every clinical session; 80% full-time faculty members who are American Board of Orthodontics (ABO) certified; a craniofacial faculty member; 4 residents/graduate students per each faculty member who covers a clinical session; resident/graduate student exposure to a wide range of treatment modalities and appliances; approximately 70 new case starts per resident/graduate student (50%-60% of patients who are started are debonded by the starting resident/graduate student); patients with craniofacial anomalies and orthognathic surgery patients should be started by each resident/graduate student; 1.5 operatory chairs per resident or graduate student; 1 dental assistant per 4 residents/graduate students; 1 laboratory person; 1 receptionist/secretary per 4 residents; 100% of residents/graduate students successfully completing ABO written examination upon graduation; 60% of residents/graduate students obtaining ABO certification within 5 years of graduation; 50% of residents/graduate students presenting at national meetings would be ideal; and 50% of living alumni contributing financially to the department during the past 5 years. CONCLUSIONS: Based on the responses from the majority of the US orthodontic residency programs, this study has identified certain features that educators feel are ideal for an excellent orthodontic program.

Changes in Central Macular Thickness and Retinal Nerve Fiber Layer Thickness in Eyes with Vogt-Koyanagi-Harada Disease: A 2-Year Follow-Up Study.

PURPOSE: To investigate the central macular thickness (CMT) and retinal nerve fiber layer (RNFL) thickness in eyes with Vogt-Koyanagi-Harada (VKH) disease associated with optic disc swelling and serous retinal detachment through a 24-month follow-up period. METHODS: We prospectively investigated 28 eyes of 14 treatment-naïve patients with acute VKH disease associated with optic disc swelling and serous retinal detachment and 30 eyes of 15 normal individuals to compare changes in the CMT and average RNFL thickness. RESULTS: The CMT was significantly lower in the eyes of the VKH group at 12 and 24 months. The RNFL thickness was significantly higher in the eyes of the VKH group at the initial visit and at the 6- and 12-month follow-up visits, but no significant difference was found between the VKH group and control group at the 24-month follow-up visit. CONCLUSION: Significant changes in the CMT and RNFL thickness in the eyes with VKH disease were observed during the 24-month follow-up period. When diagnosing or monitoring diseases including glaucoma and neuro-ophthalmic diseases that affect the retinal thickness in patients with VKH disease, we recommend considering longitudinal changes in the retinal thickness.

The Invisalign Appliance Could Be an Effective Modality for Treating Overbite Malocclusions Within a Mild to Moderate Range.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: Management of overbite with the Invisalign appliance. Khosravi R, Cohanim B, Hujoel P, Daher S, Neal M, Liu W, Huang G. Am J Orthod Dentofacial Orthop 2017;151:691-9. SOURCE OF FUNDING: This study was supported by the University of Washington Orthodontic Alumni Association TYPE OF STUDY/DESIGN: A retrospective study.

Self-ligating Brackets May Not Have Clinical Advantages Over Conventional Brackets for the Periodontal Health of Adolescent Orthodontic Patients.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: The effect of bracket ligation on the periodontal status of adolescents undergoing orthodontic treatment. A systematic review and meta-analysis. Arnold S, Koletsi D, Patcas R, Eliades T. J Dent 2016; 54:13-24. SOURCE OF FUNDING: The authors did not report any funding source information for the study TYPE OF STUDY/DESIGN: Systematic review with meta-analysis of data.

Presence of gingivitis and periodontitis significantly increases hospital charges in patients undergoing heart valve surgery.

OBJECTIVE: To examine the prevalence and impact of gingivitis and periodontitis in patients having heart valve surgical procedures. METHODS: Nationwide Inpatient Sample for the years 2004-2010 was used. All patients who had heart valve surgical procedures were selected. Prevalence of gingivitis/periodontitis was examined in these patients. Impact of gingivitis/periodontitis on hospital charges, length of stay, and infectious complications was examined. RESULTS: 596,190 patients had heart valve surgical procedures. Gingivitis/periodontitis was present in 0.2 percent. Outcomes included: median hospital charges ($175,418 with gingivitis/ periodontitis versus $149,353 without gingivitis/periodontitis) and median length of stay (14 days with gingivitis/periodontitis versus 8 days without gingivitis/periodontitis). After adjusting for the effects of patient- and hospital-level confounding factors, hospital charges and length of stay were significantly higher (p < 0.001) in those with gingivitis/periodontitis compared to their counterparts. Further, patients with gingivitis/periodontitis had significantly higher odds for having bacterial infections (OR = 3.41, 95% CI = 2.33-4.98, p < 0.0001) when compared to those without gingivitis/periodontitis. CONCLUSION: Presence of gingivitis and periodontitis is associated with higher risk for bacterial infections and significant hospital resource utilization.

Sectoral retinal nerve fiber layer thinning in branch retinal vein occlusion.

PURPOSE: To analyze longitudinal changes in the retinal nerve fiber layer (RNFL) thickness in branch retinal vein occlusion (BRVO) using optical coherence tomography. METHODS: The authors prospectively analyzed 20 eyes in 20 patients diagnosed with branch retinal vein occlusion (BRVO) and followed for more than 1 year. The RNFL thickness of the normal and occluded eyes was measured at the time of diagnosis and at 1, 3, 6, and 12 months. The authors compared the changes in the occluded eye over the follow-up and the differences between two eyes at each time. They also analyzed the area opposite the occluded area. RESULTS: The thickness of the RNFL in BRVO was significantly reduced at 1, 3, 6, and 12 months (P = 0.005 at 1 month and P = 0.001 at 3, 6, and 12 months) compared with initial thickness. The RNFL thickness at 3 months did not differ significantly between 2 eyes, whereas at 6 months and 12 months, significant (P = 0.032, P = 0.002, respectively.) thinning was observed in the occluded eye. Analysis of the area opposite that of BRVO revealed no significant (P > 0.05) change during the follow-up and no difference between the 2 eyes. CONCLUSION: There was a significant decrease in RNFL thickness over time in BRVO and significant thinning at 6 months compared with the normal eye. Retinal nerve fiber layer thinning needs to be differentiated from glaucoma or systemic disease, but it should be considered the natural course after BRVO.

Enhancing Craniofacial Bone Reconstruction with Clinically Applicable 3D Bioprinted Constructs.

Medical imaging and 3D bioprinting can be used to create patient-specific bone scaffolds with complex shapes and controlled inner architectures. This study investigated the effectiveness of a biomimetic approach to scaffold design by employing geometric control. The biomimetic scaffold with a dense external layer showed improved bone regeneration compared to the control scaffold. New bone filled the defected region in the biomimetic scaffolds, while the control scaffolds only presented new bone at the boundary. Histological examination also shows effective bone regeneration in the biomimetic scaffolds, while fibrotic tissue ingrowth is observed in the control scaffolds. These findings suggest that the biomimetic bone scaffold, designed to minimize competition for fibrotic tissue formation in the bony defect, can enhance bone regeneration. This study underscores the notion that patient-specific anatomy can be accurately translated into a 3D bioprinting strategy through medical imaging, leading to the fabrication of constructs with significant clinical relevance.

Pharmacotherapies to prevent epidural fibrosis after laminectomy: a systematic review of in vitro and in vivo animal models.

BACKGROUND CONTEXT: Excessive production of epidural fibrosis in the nerve root can be a pain source after laminectomy. Pharmacotherapy is a minimally invasive treatment option to attenuate epidural fibrosis by suppressing proliferation and activation of fibroblasts, inflammation, and angiogenesis, and inducing apoptosis. PURPOSE: We reviewed and tabulated pharmaceuticals with their respective signaling axes implicated in reducing epidural fibrosis. Additionally, we summarized current literature for the feasibility of novel biologics and microRNA to lessen epidural fibrosis. STUDY DESIGN/SETTING: Systematic Review. METHODS: According to the PRISMA guidelines, we systematically reviewed the literature in October 2022. The exclusion criteria included duplicates, nonrelevant articles, and insufficient detail of drug mechanism. RESULTS: We obtained a total of 2,499 articles from PubMed and Embase databases. After screening the articles, 74 articles were finally selected for the systematic review and classified based on the functions of drugs and microRNAs which included inhibition of fibroblast proliferation and activation, pro-apoptosis, anti-inflammation, and antiangiogenesis. In addition, we summarized various pathways to prevent epidural fibrosis. CONCLUSION: This study allows a comprehensive review of pharmacotherapies to prevent epidural fibrosis during laminectomy. CLINICAL SIGNIFICANCE: We expect that our review would enable researchers and clinicians to better understand the mechanism of anti-fibrosis drugs for the clinical application of epidural fibrosis therapies.

Anterior openbite malocclusion in adults.

OBJECTIVES: To investigate stability and satisfaction in adult anterior open bite (AOB) patients at least 9 months post-treatment, as well as patient and practitioner factors that may be associated with stability and satisfaction. MATERIALS AND METHODS: Practitioners and their adult AOB patients were recruited through the National Dental Practice-Based Research Network. Data on patient and practitioner characteristics, treatment recommendations and factors were previously collected. Treatment stability was determined by assessing post-treatment intraoral photographs. Patient satisfaction was determined from post-treatment questionnaires. Treatment was categorized into aligners, fixed appliances, temporary anchorage devices, and orthognathic surgery. Extractions were also investigated. Retention type was categorized into vacuum-formed, Hawley-style, or bonded retainers, and regimens were classified as full-time or part-time wear. RESULTS: Retention data collected from 112 patients had a mean post-treatment time of 1.21 years. There were no statistically significant differences in stability between treatment groups. Depending on whether a qualitative index or a millimetric measure was employed, stability ranged from 65% to 89%. Extractions and less initial lower incisor proclination were associated with higher stability in patients treated with fixed appliances only. High satisfaction was reported by patients at retention. There were no clear differences in stability or satisfaction among retention types or regimens. CONCLUSIONS: The stability of adult AOB orthodontic treatment was high, regardless of treatment or retainer modality. Satisfaction in adult AOB patients was high, regardless of retention type or regimen.

Polydopamine functionalized VEGF gene-activated 3D printed scaffolds for bone regeneration.

Bone is a highly vascularized organ and the formation of new blood vessels is essential to regenerate large critical bone defects. In this study, polylactic acid (PLA) scaffolds of 20-80% infill were three-dimensionally (3D) printed using a fused deposition modeling based 3D printer. The PLA scaffolds were coated with polydopamine (PDA) and then were surface-functionalized with polyethyleneimine (PEI) and VEGF-encoding plasmid DNA (pVEGF) nanoplexes (PLA-PDA-PEI-pVEGF). The PLA-PDA-PEI-pVEGF scaffolds with 40% infill demonstrated higher encapsulation efficiency and sustained release of pVEGF than scaffolds with 20, 60 and 80% infill and were therefore used for in vitro and in vivo studies. The PLA-PDA-PEI-pVEGF increased the translation and secretion of VEGF and BMP-2. The PLA-PDA-PEI-pVEGF also yielded a 2- and 4.5-fold change in VEGF and osteocalcin gene expression in vitro, respectively. A tube formation assay using human umbilical vascular endothelial cells (HUVECs) showed a significant increase in tube length when exposed to the PLA-PDA-PEI-pVEGF scaffold, in comparison to PLA and PLA-PDA scaffolds. The PLA-PDA-PEI-pVEGF scaffold in an in vivo rat calvarial critical bone defect model demonstrated 1.6-fold higher new bone formation compared to the PLA-PDA scaffold. H&E and Masson's trichrome staining of bone sections also revealed that the PLA-PDA-PEI-pVEGF scaffold facilitated the formation of more blood vessels in the newly formed bone compared to the PLA and PLA-PDA scaffold groups. Thus, PLA-PDA-PEI-pVEGF might be a potential 3D printed gene activated scaffold for bone regeneration in clinical situations.

Nonviral Gene Delivery Embedded in Biomimetically Mineralized Matrices for Bone Tissue Engineering.

Research in bone tissue engineering aims to design materials that are effective at generating bone without causing significant side effects. The osteogenic potential of combining matrices and protein growth factors has been well documented, however, improvements are necessary to achieve optimal therapeutic benefits upon clinical translation. In this article, rat calvarial defects were treated with gene-activated matrices (GAMs). The GAMs used were collagen sponges mineralized with a simulated body fluid (SBF) containing a nonviral gene delivery system. Both in vitro and in vivo studies were performed to determine the optimal mode of gene delivery. After 6 weeks, the defects were extracted to assess bone formation and tissue quality through histological and microcomputed tomography analyses. The optimal GAM consisted of a collagen sponge with polyethylenimine plasmid DNA (PEI-pDNA) complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared with 19% for control samples. A follow-up in vivo study was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was five parts of pBMP-2 to three parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) can optimally improve bone regeneration outcomes. Impact statement Bone tissue engineering has explored both nonviral gene delivery and the concept of biomimetic mineralization. In this study, we combined these two concepts to further enhance bone regeneration outcomes. We demonstrated that embedding polyethylenimine (PEI)-based gene delivery within a mineral layer formed from simulated body fluid (SBF) immersion can increase bone formation rates. We also demonstrated that the ratio of growth factors utilized for matrix fabrication can impact the amount of bone formed in the defect site. This research highlights a combined approach using SBF and nonviral gene delivery both in vitro and in vivo and prepares the way for future optimization of synthetic gene activated matrices.

Multidisciplinary care for a patient with syndromic craniosynostosis: A case report with 20 years of special care.

AIM: The functional and structural complexities accompanying syndromic craniosynostosis make dental care for these patients particularly challenging. We report a case of long-term care for a syndromic craniosynostosis patient. The objective of this report is to introduce special care guidance and clinical recommendation, so that oral health care providers, as key members of a multidisciplinary care team, can provide optimal diagnosis, treatment, and management for the patient with syndromic craniosynostosis. CASE REPORT: The patient of this case report had a medical history of syndromic craniosynostosis involving multiple comorbidities. Over the past 20 years, a multidisciplinary care team has successfully treated the patient. Dental and medical procedures that the patient has received include cranial surgeries, prophylactic dental care, caries control, growth hormone therapy, comprehensive orthodontic treatment in conjunction with orthognathic surgeries, and plastic surgery. CONCLUSION: Oral health care providers can play essential roles in multidisciplinary care for patients with craniosynostosis by understanding the patients' unique oral health conditions and dentofacial deformities. To provide optimal oral health care in a multidisciplinary team, clear communication between the members of the care team is crucial.

Effects of calcium concentration on nonviral gene delivery to bone marrow-derived stem cells.

BACKGROUND: Calcium ions (Ca2+ ) influence natural bone healing, and calcium is frequently used in bone tissue engineering scaffolds and cements. Scaffolds can also incorporate gene delivery systems to further promote osteoblast differentiation. Thus, our goal was to identify if Ca2+ concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene-activated scaffolds designed to promote bone growth. METHODS: Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in media with Ca2+ concentrations ranging from 0 to 20 mM and transfected with polyethyleneimine-plasmid DNA (PEI-pDNA) complexes. Cell viability and transfection efficiency were determined using MTS assays and flow cytometry, respectively. PEI-pDNA complex localization in BMSCs was assessed using fluorescence microscopy. To determine BMSC differentiation, messenger RNA (mRNA) for osteocalcin and CBFA1 was quantified using real time-polymerase chain reaction (PCR). Calcium deposition was qualitatively assessed after three and 14 days using Alizarin Red staining. RESULT: Our results indicate that Ca2+ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI-pDNA complexes in terms of cell viability and transfection efficiency. A Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP-2). CONCLUSION: Ca2+ at a 10 mM concentration can significantly reduce toxicity and enhance transfection efficiency when combined with PEI-pDNA complexes, and this combination can be specifically applied to further enhance the differentiation of BMSCs by using the combination of polyethyleneimine-plasmid bone morphogenetic protein 2 (PEI-pBMP-2) and 10 mM Ca2+ as compared with PEI-pBMP-2 alone.