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Central giant cell granuloma of the jaw (CGCJ) can be locally aggressive and result in facial and dental deformity. A child with CGCJ was treated surgically and with denosumab with a response but life-threatening toxicity. Imatinib, a tyrosine kinase inhibitor, was prescribed based on clinical similarities between CGCJ and cherubism, for which Imatinib has been effective. Within 2 months, a computed tomographic scan showed significant ossification, which increased over the following 8 months. This case suggests that tyrosine kinase inhibitors may be an effective option, and one with limited toxicity, for CGCJ., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

Mucoepidermoid carcinoma (MEC) is a life-threatening salivary gland cancer that is driven primarily by a transcriptional coactivator fusion composed of cyclic AMP-regulated transcriptional coactivator 1 (CRTC1) and mastermind-like 2 (MAML2). The mechanisms by which the chimeric CRTC1/MAML2 (C1/M2) oncoprotein rewires gene expression programs that promote tumorigenesis remain poorly understood. Here, we show that C1/M2 induces transcriptional activation of the non-canonical peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) splice variant PGC-1α4, which regulates peroxisome proliferator-activated receptor gamma (PPARγ)-mediated insulin-like growth factor 1 (IGF-1) expression. This mitogenic transcriptional circuitry is consistent across cell lines and primary tumors. C1/M2-positive tumors exhibit IGF-1 pathway activation, and small-molecule drug screens reveal that tumor cells harboring the fusion gene are selectively sensitive to IGF-1 receptor (IGF-1R) inhibition. Furthermore, this dependence on autocrine regulation of IGF-1 transcription renders MEC cells susceptible to PPARγ inhibition with inverse agonists. These results yield insights into the aberrant coregulatory functions of C1/M2 and identify a specific vulnerability that can be exploited for precision therapy., Competing Interests: Declaration of interests A.L.A. is a GAB member and paid consultant for LG Chem Life Sciences Innovation Center., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The use of diverse terminology may lead to inconsistent diagnosis and subsequent mistreatment of lesions within the proliferative verrucous leukoplakia (PVL) spectrum. The objectives of this study were: (a) to measure inter-observer variability between a variety of pathologists diagnosing PVL lesions; and (b) to evaluate the impact of diverse terminologies on understanding, interpretation, and subsequent treatment planning by oral and maxillofacial surgeons (OMFS). Six oral pathologists (OP) and six head and neck pathologists (HNP) reviewed 40 digitally scanned slides of PVL-type lesions. Inter-observer agreement on diagnoses was evaluated by Fleiss' kappa analysis. The most commonly used diagnostic terminologies were sent to ten OMFS to evaluate their resulting interpretations and potential follow-up treatment approaches. The overall means of the surgeons' responses were compared by Student t test. There was poor inter-observer agreement between pathologists on the diagnosis of PVL lesions (κ = 0.270), although there was good agreement (κ = 0.650) when diagnosing frankly malignant lesions. The lowest agreement was in diagnosing verrucous hyperplasia (VH) with/without dysplasia, atypical epithelial proliferation (AEP), and verrucous carcinoma (VC). The OMFS showed the lowest agreement on identical categories of non-malignant diagnoses, specifically VH and AEP. This study demonstrates a lack of standardized terminology and diagnostic criteria for the spectrum of PVL lesions. We recommend adopting standardized criteria and terminology, proposed and established by an expert panel white paper, to assist pathologists and clinicians in uniformly diagnosing and managing PVL spectrum lesions.

Genome-wide association studies (GWAS) have identified Optineurin (OPTN) as genetically linked to Paget's disease of the bone (PDB), a chronic debilitating bone remodeling disorder characterized by localized areas of increased bone resorption and abnormal bone remodeling. However, only ~10% of mouse models with a mutation in Optn develop PDB, thus hindering the mechanistic understanding of the OPTN-PDB axis. Here, we reveal that 100% of aged Optn global knockout (Optn -/- ) mice recapitulate the key clinical features observed in PDB patients, including polyostotic osteolytic lesions, mixed-phase lesions, and increased serum levels of alkaline phosphatase (ALP). Differentiation of primary osteoclasts ex vivo revealed that the absence of Optn resulted in an increased osteoclastogenesis. Mechanistically, Optn-deficient osteoclasts displayed a significantly decreased type I interferon (IFN) signature, resulting from both defective production of IFNβ and impaired signaling via the IFNα/βR, which acts as a negative feedback loop for osteoclastogenesis and survival. These data highlight the dual roles of OPTN in the type I IFN response to restrain osteoclast activation and bone resorption, offering a novel therapeutic target for PDB. Therefore, our study describes a novel and essential mouse model for PDB and define a key role for OPTN in osteoclast differentiation.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that affects the skin, brain, kidneys, and other organ systems. It may exhibit a wide spectrum of clinical manifestations. Desmoplastic fibroma (DF) of the jaw is a rare benign myofibroblastic neoplasm. Less than 10 cases of DF associated with TSC have been published previously. We report a new case of a maxillary DF in a 12-year-old girl with TSC. The presentation, diagnostic process, and management of this case are discussed, and the literature is reviewed for the additional cases of DF associated with TSC; 7 previously reported cases are summarized. Small sample size limits conclusions, but there may be differences in the presentations of DF of the jaws in patients with TSC vs those in the general population. DF of the jaws may be a manifestation of TSC, and the authors propose surveillance panoramic radiographs every 2 to 3 years in patients with TSC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to perform a systematic analysis of the nicotinamide adenine dinucleotide phosphate (NAD[P])-dependent steroid dehydrogenase-like (NSDHL) gene in cases of oral verruciform xanthoma (VX) and to test for the presence of mutations associated with congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome., Study Design: DNA was extracted from archived paraffin-embedded tissue of oral VX and control cases. Polymerase chain reaction (PCR) was then used to screen exons 4 and 6 of the NSDHL gene for the presence of 4 known germline mutations associated with CHILD syndrome and 1 somatic mutation previously identified in VX lesions with no known association with CHILD syndrome., Results: Of the 16 oral VX tissue samples, 8 (50%) had known missense mutations associated with CHILD syndrome. Furthermore, 2 of these 8 tissue samples also had an additional missense mutation previously identified in cutaneous VX lesions. No mutations of exons 4 and 6 were found in the 5 negative control tissue samples., Conclusions: NSDHL gene mutations associated with CHILD syndrome are common in sporadic oral VX cases, suggesting that these mutations confer a greater risk for the development of epithelial barrier defects that promote recurrent oral VX lesions and the potential for direct germline transmission of oral VX susceptibility., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to identify the type and distribution of CD4+ and CD8+ T lymphocytes in oral mucosal specimens to potentially distinguish between underlying alterations or patterns in oral epithelial dysplasia and oral lichen planus., Study Design: This pilot study included 10 archived tissue samples that were received at the University of North Carolina Oral and Maxillofacial Pathology Laboratory and were diagnosed as oral lichen planus and moderate to severe epithelial dysplasia. Dual staining with CD4 and CD8 antibodies was carried out on each case. Slides were scanned in the Aperio ScanScope FL (Leica Biosystems, Wetzlar, Germany) and archived. Histomorphometric analysis was performed to detect inflammatory cells expressing CD4 and CD8 biomarkers in the epithelial and connective tissue regions., Results: No differences were found in the amount and ratio of CD4+/CD8+ lymphocytes among the 3 groups analyzed; however, the intraepithelial CD8+ lymphocyte distribution was strikingly different between lichen planus and moderate to severe epithelial dysplasia., Conclusions: The localization of CD8+ cells can be potentially useful as an adjunctive diagnostic procedure to distinguish oral epithelial dysplasia from other inflammatory entities, such as lichen planus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease affecting exocrine glands, thereby causing dry mouth and eyes (sicca). Our objective was to determine the expression of pSS pathogenic biomarker MMP9 and its putative transcription factors ETS1 and LEF1, in labial salivary glands of pSS patients., Methods: Sicca patients were assigned to three groups based on focus score (FS): non-pSS sicca (i.e., GR1 [FS = 0] and GR2 [0 < FS < 1]) and pSS (i.e., GR3 [FS ≥ 1]). We determined the mRNA and protein expression of MMP9, ETS1, and LEF1 in salivary gland biopsies. Also, ETS1-CD4 and LEF1-CD4 co-expression analyses were performed., Results: The mRNA expression of MMP9, ETS1, and LEF1 was upregulated in GR3 compared to GR1 (p < 0.01). Most GR3 salivary gland areas had moderate to high MMP9, ETS1, and LEF1 protein expression compared to GR1 and GR2. Further, ETS1-CD4 and LEF1-CD4 dual staining demonstrated that both salivary gland epithelial cells and lymphocytic infiltrates had increased levels of ETS1 and LEF1. Moreover, there was a strong correlation between ETS1(+)-CD4(-) and LEF1(+)-CD4(-) cells., Conclusion: These results suggest, for the first time, a concerted increase in ETS1 and LEF1 expression in salivary gland epithelial cells of pSS patients that is reflective of the etiopathogenesis of pSS., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Objectives: The aims of this study were to determine whether lesion features appear differently on panoramic radiography (PAN) and cone beam computed tomography (CBCT), and whether the use of CBCT affects diagnostic accuracy and observers' confidence in comparison with PAN., Study Design: Three oral and maxillofacial radiologists reviewed 33 sets of PAN images and CBCT volumes of biopsy-proven lesions. They described 12 different lesion features and provided up to 3 ranked differential diagnoses, as well as their confidence with respect to those diagnoses. Their confidence was weighted by the rank at which the correct diagnosis was provided., Results: Odds ratios (ORs) were statistically significant for border definition (OR = 5.45; P = .004), continuity of border cortication (OR = 0.34; P = .035), effect on neurovascular canals (OR = 6.38; P = .043), expansion (OR = 18.56; P < .001), cortical thinning (OR = 30.22; P < .001), and cortical destruction (OR = 9.80; P < .001). There was no association between the 2 modalities and the rank at which the correct differential diagnoses were made or the observers' weighted confidence., Conclusions: Before acquiring a CBCT scan to aid in the diagnosis of an intraosseous lesion, clinicians should consider the diagnostic information that is expected to be gained. In this study, although there were differences between PAN and CBCT with respect to some lesion features, CBCT did not help improve diagnostic accuracy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: We report here the 10-year experience with oral hairy leukoplakia (OHL) at the Division of Oral and Maxillofacial Pathology at the University of North Carolina at Chapel Hill, NC, USA., Study Design: All the associated hematoxylin and eosin and Epstein-Barr virus encoding region in situ hybridization slides of OHL cases between January 1, 2008, and February 1, 2017, were retrieved and reviewed. Collected demographic characteristics, clinical presentation, medical and social histories were reviewed and reported., Results: Six OHL cases with confirmed in situ hybridization showed predilection for the lateral tongue. The study included 3 females and 3 males (mean age 50.5 years; age range 29-70 years). One patient had known HIV-positive status before biopsy was performed. Three patients had reported a history of heavy smoking. Other medical conditions reported were history of breast cancer, a long history of corticosteroid inhaler use for asthma treatment, high cholesterol, diabetes, and hypertension., Conclusions: The findings of this study indicate the need to include OHL as a potential entity in the differential diagnosis of leukoplakic tongue lesions, regardless of the patient's HIV status. In addition, the presence of OHL in the patient requires investigation of various explanations for EBV infection, including immunosuppression caused by HIV infection or chronic steroid use., (Published by Elsevier Inc.)

The formation of biofilms by Candida and the increasing resistance of Candida species to antifungals contribute to the high recurrence rates of denture stomatitis. This increase has stimulated an interest in antimicrobial photodynamic therapy (aPDT) as an alternative treatment. We examined the photoactivity of the porphyrin-based photosensitizer, TMP-1363, against biofilms of C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, and the effect of the combined use of miconazole and aPDT. Biofilms of three American Type Culture Collection (ATCC) strains and four clinical isolates developed on poly(methyl methacrylate) (PMMA) disks, were incubated with miconazole, followed by treatment with TMP-1363 for 30 min at 37°C. The plates were exposed to broadband visible light at a distance of 10 cm to the plate, for 30 min (irradiance at the surface of the plate: 32.5 mW/cm2). The metabolic activity of the biofilms was measured by the XTT assay. ATCC strains and C. glabrata 7531/06 were not sensitive to TMP-aPDT, whereas the metabolic activities of the remaining three clinical isolates were reduced to 64.2 ± 5.5% of controls. Miconazole at 25 μg/ml decreased the viability of all strains except the ATTCC strain C. albicans MYA274; however its combination with aPDT was effective against this strain, suggesting a synergistic interaction. Effects of miconazole and aPDT on C. albicans MYA 2732, C. albicans 6122/06 were additive. With C. tropicalis and C. parapsilosis, the combined treatment had a higher, but not entirely additive, cytotoxic effect. The combined use of miconazole and TMP-aPDT is advantageous in the treatment of biofilms of a number of Candida species and strains, but not all. The molecular basis of this differential response is not known.

Objective: To delineate the features of carcinoma cuniculatum (CC), emphasizing potential management errors., Study Design: A retrospective study examined archival cases of CC. An analysis of clinical, microscopic, and management parameters was performed., Results: Ten cases were identified, and their clinical and microscopic features were summarized. CC exhibits a sessile pink/red mildly papillary surface. Histologically, CC presents a tortuous invasive component with a more subtle papillary appearance than verrucous carcinoma., Conclusions: CC is an uncommon variant of squamous cell carcinoma distinct from verrucous carcinoma. Diagnostic delays result from misinterpretation of superficial samples or lack of awareness of the entity. Bland cytology and unusual architecture result in underdiagnosis of CC without clinicopathologic correlation. Clinicians should submit multiple deep samples of lesions displaying a cobblestone-like surface and provide a clinical photograph to the pathologist. Pathologists can avoid underdiagnosis by thorough sampling of biopsies and requesting additional tissue as needed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To determine whether a collagen scaffold could provide an environment for mesenchymal stem cell (MSC)-related bone repair of critical-size bone defects in rat calvaria., Materials and Methods: Craniotomy defects were created in 28 adult Sprague-Dawley rats. Two additional rats were used as MSC donors by means of femoral bone marrow lavage and culture. The rats were randomly divided into four groups: (1) empty/no graft; (2) collagen scaffold (matrix)+saline; (3) matrix+MSCs; (4) matrix+bone morphogenetic protein. The animals were euthanized 28 days after surgery. Microcomputed tomographic reconstructions were obtained to measure bone fill. The specimens were processed for histologic examination, and the total defect and bone fill areas were measured., Results: Mean bone fill (± standard deviation) of 9.25%±10.82%, 19.07%±17.38%, 44.21%±3.93%, and 66.06%±15.08%, respectively, was observed for the four groups; the differences were statistically significant. Bone repair was statistically significant for groups 3 and 4. No significant difference was seen for bone repair between groups 1 and 2 or between groups 3 and 4. Bone formation differed significantly across the four groups. Statistically significant changes in radiodensity were observed between groups 1 and 3, groups 1 and 4, and groups 2 and 4. Significant differences were not observed between groups 1 and 2, groups 2 and 3, or groups 3 and 4., Conclusion: After grafting of adult MSCs adherent within a collagen matrix, repair of bone was significant. Expanded three-dimensional collagen represents a radiolucent, resorbable, biocompatible scaffold that is capable of supporting MSC repair of bone.

Objectives: Oral mucositis caused by cancer chemotherapy can result in significant clinical complications. There is a strategic need to accelerate the delineation of the pathobiology. This proof-of-principle study was designed to demonstrate the feasibility of studying archived oral mucosal specimens to further delineate oral mucositis pathobiology., Materials and Methods: Twenty-nine formalin-fixed and paraffin-embedded tissue blocks of 25-year-old oral mucosa autopsy specimens from cancer chemotherapy patients were studied. Standardized technology was utilized, including RNA isolation and amplification, array hybridization, and gene expression analysis., Results: A predominance of DNA damage in buccal mucosal basal keratinocytes was observed. Data comparing basal cells from buccal vs. gingival mucosa identified differential gene expression of host responses in relation to pathways relevant to oral mucositis pathogenesis, including responses to cancer-associated inflammation., Conclusions: This proof-of-principle study demonstrated that archived oral mucosal specimens may be a potentially valuable resource for the study of oral mucositis in cancer patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To evaluate the type and prevalence of incidental findings from cone beam computed tomography (CBCT) of the maxillofacial region. Findings are divided into those that require (i) intervention/referral, (ii) monitoring, and (iii) no further evaluation., Methods: Three hundred consecutive CBCT scans conducted in the University of North Carolina School of Dentistry Oral and Maxillofacial Radiology Clinic from January 1 to August 31, 2008 were retrospectively reviewed. Findings were categorized into airway, soft tissue calcifications, bone, temporomandibular joint (TMJ), endodontic, dental developmental, and pathological findings., Results: A total of 272 scans revealed 881 incidental findings (3.2 findings/scan). The most prevalent was airway findings (35%) followed by soft tissue calcifications (20%), bone (17.5%), TMJ (15.4%), endodontic (11.3%), dental developmental (0.7%), and pathological (0.1%). 16.1% required intervention/referral, 15.6% required monitoring, and the remainder (68.3%) required neither., Conclusion: This study underscores the need to thoroughly examine all CBCT volumes for clinically significant findings within and beyond the region of interest., (© 2011 John Wiley & Sons A/S.)

Seven expansile jaw lesions in patients ranging from 7 to 63 years are presented to illustrate diagnostic and management issues pertaining to cases ultimately proven to be gnathic osteosarcoma (GO). Six of the cases in our series were low-grade osteoblastic and one high-grade chondroblastic. None of our cases exhibited the characteristic "sunburst" radiopaque appearance described for GO. All of our cases displayed cortical expansion and one showed development of diastema. Two occurred in the maxilla and five in the mandible. Two of the patients with mandibular lesions presented initially with pain; all other patients were asymptomatic. Lack of pain resulted in a delay in diagnosis due to postponement of consultation or biopsy. Two cases underwent initial shallow non-representative biopsies, requiring a second biopsy for definitive diagnosis, further delaying treatment. Those biopsies were initially interpreted as pyogenic granuloma and peripheral ossifying fibroma, respectively. GO should always be considered in the differential diagnosis of expansile jaw lesions. Bone biopsies of lesions exhibiting pain and expansion of cortical plates should include medullary bone in order to minimize sampling error. In addition, all rapidly growing or painful exophytic bone lesions, and presumed soft tissue lesions that may involve underlying bone, should be examined histopathologically, and receive clinical and radiographic follow-up until complete resolution or healing is evident, regardless of the diagnosis. Based on the positive outcomes of the patients in our series, the prognosis of GO appears to be relatively favorable when compared to other sarcomas and osteosarcomas of long bones.

A 14-year-old Caucasian female was referred by her orthodontist with a non-expansile radiolucent lesion associated with impacted tooth #31.

It is widely accepted that implant surface factors affect the quality of the bone-to-implant interface. Recent additional treatments superimposed on moderately rough cpTitanium surface provide further enhancement of bone-to-implant contact. The aim of this study was to compare osteoinductive and bone-specific gene expression in cells adherent to titanium dioxide-grit blasted (TiO2) versus TiO2 grit blasted and HF treated (TiO2/HF) cpTitanium implant surfaces. MC3T3-E1 cells were grown in osteogenic supplements on the titanium disk surfaces for 1-14 days. Real-time PCR was used to measure RUNX-2, Osterix, and bone sialoprotein (BSP) mRNA levels. Implants were placed in rat tibia and, following harvesting at 1-7 days after placement, real-time PCR was used to measure RUNX-2, alkaline phosphatase (ALP), and BSP mRNA levels in implant adherent cells. In cell culture, RUNX-2 and Osterix levels were significantly increased (p<0.05) on the TiO2/HF surfaces as compared to the TiO2 and smooth surfaces through the cultural period, while BSP expression was elevated on both TiO2 and TiO2/HF surfaces when compared to a machined surface control. In cells adherent to implants retrieved from rat tibia, RUNX-2 mRNA levels were 2-fold and 8-fold greater on the TiO2/HF surfaces at 1-3 and 7 days following implantation. This was paralleled by significantly greater levels of ALP at 3 and 7 days and BSP mRNA at 7 days following implantation. As a marker of osteoinduction, the increased levels of RUNX-2 in cells adherent to the TiO2/HF surfaces suggest that the additional HF treatment of the TiO2 grit blasted surface results in surface properties that support adherent cell osteoinduction. In vivo assessments of implant adherent cell phenotypes provide further insight into the mechanisms affecting alloplast-tissue interactions.

Background: Bisphosphonates are used widely to manage skeletal disorders resulting from malignancies that destroy bone and from some metabolic bone diseases. A strong association between bisphosphonate treatment and the appearance of painful exposed nonvital bone in the mandible and maxilla after oral surgery has been reported in the last decade. Extensive reviews have appeared in the dental literature regarding bisphosphonate-related osteonecrosis of the jaws (BRONJ), including protocols for diagnosis, management and diagnostic imaging for early detection; feature definition; and determination of extent of the disease., Case Description: The authors provide three case reports to show the contrast in treatment outcomes and morbidity in patients with BRONJ. The cases involved diagnostic imaging modalities commonly used in the practice of dentistry: panoramic radiography and cone-beam volumetric computed tomography., Clinical Implications: These case reports demonstrate the usefulness of dental diagnostic imaging in the detection and management of BRONJ, corroborate the increasing number of reports regarding high levels of morbidity associated with various BRONJ treatments, and underscore the danger of performing invasive dental procedures for patients receiving bisphosphonate therapy.

Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid. The aim of this study was to compare the osteogenic potential of predifferentiated and undifferentiated bone marrow-derived, culture-expanded hMSCs adherent to synthetic HA/TCP (60%/40%) following subcutaneous engraftment in severe combined immunodeficiency (SCID) mice. During the final 3 days of culture, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics or media containing 25-mM calcium supplementation with vitamin D and ascorbic acid. Four weeks following implantation in SCID mice, scoring analysis of bone formation within the cubes revealed the absence of bone formation in unloaded cubes. Bone formation compared by a qualitative bone index was 7.23% for undifferentiated cells compared to 5.20% for differentiated cells. Minimal resorption was observed at this early time point. In this ectopic model, predifferentiation using a combination of vitamin D and ascorbic acid failed to increase subsequent bone formation by implanted cells. Following implantation of hMSCs adherent to an osteoconductive scaffold, host factors may contribute dominant osteoinductive signals or impose inhibitory signals to control the fate of the implanted cell. Predifferentiation strategies require confirmation in vivo.