Elevated nutrient availability enhances chondrocyte metabolism and biosynthesis in tissue-engineered cartilage.

OBJECTIVE: Chondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in hypoxia-inducible factor 1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. METHODS: Primary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4-16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). RESULTS: Increasing media volumes resulted in higher accumulation of cartilaginous ECM (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the tricarboxylic acid (TCA) cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. CONCLUSIONS: Elevated media availability upregulates ECM synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in ECM accumulation.

13C Metabolic Flux Analysis in Chondrocytes Reveals a Novel Switch in Metabolic Phenotype.

Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through 13C-metabolic flux analysis (13C-MFA), as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-13C] glucose labeling experiments were combined with a tissue-specific metabolic network model to carry out 13C-MFA under varying levels of nutrient availability. 13C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic-anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow toward the tricarboxylic acid cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect ("inverse Crabtree effect"). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes-a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.

Effect of nutrient metabolism on cartilaginous tissue formation.

A major shortcoming in cartilage tissue engineering is the low biosynthetic response of chondrocytes. While different strategies have been investigated, a novel approach may be to control nutrient metabolism. Although known for their anaerobic metabolism, chondrocytes are more synthetically active under conditions that elicit mixed aerobic-anaerobic metabolism. Here, we postulate this metabolic switch induces HIF-1α signaling resulting in improved growth. Transition to different metabolic states can result in the pooling of metabolites, several of which can stabilize HIF-1α by interfering with PHD2. Chondrocytes cultured under increased media availability accelerated tissue deposition with the greatest effect occurring at 2 ml/106 cells. Under higher media availability, metabolism switched from anaerobic to mixed aerobic-anaerobic. Around this transition, maximal changes in PHD2 activity, HIF-1α expression, and HIF-1 target gene expression were observed. Loss-of-function studies using YC-1 confirmed the involvement of HIF-1. Lastly, targeted metabolomic studies revealed that intracellular lactate and succinate correlated with PHD2 activity. This study demonstrates that cartilaginous tissue formation can be regulated by nutrient metabolism and that this response is mediated through changes in HIF-1α signaling. By harnessing this newly identified metabolic switch, engineered cartilage implants may be developed without the need for sophisticated methods which could aid translation to the clinic.

Optimization of culture media to enhance the growth of tissue engineered cartilage.

Tissue engineering is a promising option for cartilage repair. However, several hurdles still need to be overcome to develop functional tissue constructs suitable for implantation. One of the most common challenges is the general low capacity of chondrocytes to synthesize cartilage-specific extracellular matrix (ECM). While different approaches have been explored to improve the biosynthetic response of chondrocytes, several studies have demonstrated that the nutritional environment (e.g., glucose concentration and media volume) can have a profound effect on ECM synthesis. Thus, the purpose of this study was to optimize the formulation of cell culture media to upregulate the accumulation of cartilaginous ECM constituents (i.e., proteoglycans and collagen) by chondrocytes in 3D culture. Using response surface methodology, four different media factors (basal media, media volume, glucose, and glutamine) were first screened to determine optimal media formulations. Constructs were then cultured under candidate optimal media formulations for 4 weeks and analyzed for their biochemical and structural properties. Interestingly, the maximal accumulation of proteoglycans and collagen appeared to be elicited by different media formulations. Most notably, proteoglycan accumulation was favored by high volume, low glucose-containing DMEM/F12 (1:1) media whereas collagen accumulation was favored by high volume, high glucose-containing F12 media. While high glutamine-containing media elicited increased DNA content, glutamine concentration had no apparent effect on ECM accumulation. Therefore, optimizing the nutritional environment during chondrocyte culture appears to be a promising, straight-forward approach to improve cartilaginous tissue formation. Future work will investigate the combined effects of the nutritional environment and external stimuli.

Delayed surgery in neurologically intact patients affected by thoraco-lumbar junction burst fractures: to reduce pain and improve quality of life.

BACKGROUND: This is a retrospective study on 18 patients affected by thoraco-lumbar junction burst fractures (TLJBF) A3 or A4 at computed tomography (CT) scan who referred to our hospital. To assess the surgical results in terms of pain and quality of life in a series of neurologically intact patients affected by TLJBF who underwent surgery after 3-4 months from the injury. In literature there is controversy if pain could be an indication for surgery in TLJBF and series of patients conservatively managed with success have been reported. METHODS: A retrospective study on 18 patients is reported. Patients included in this series were neurologically intact and affected by a TLJBF A3 or A4 at CT scan, the height of the burst vertebral body was >50%, spinal canal invasion was <30% and kyphosis deformity <30 degrees. Pain and quality of life were evaluated using graphic rating scale (GRS) and EuroQol (EQ-5D) scores on admission, at the clinical follow-up and in post-surgical period. RESULTS: Comparing pre- and post-operative EQ-5D, the scores had a statistically significant decrease after the operation (P<0.001) [pre-surgery EQ-5D was 2.60 (SD =0.67), post-surgery EQ-5D was 1.37 (SD =0.41)]. Also analyzing the EQ5D-VAS scores, the t-test revealed that surgery (P<0.01) improved the quality of life with statistically significance (EQ5D-VAS pre =43.89, SD =12.43 and EQ5D-VAS post =73.33, SD =10.84). Analyzing pre- and post-surgical GRS scores, the pain decreased significantly with the maximum mean difference among the 2nd and 3rd month before surgery and at 12 months after surgery (respectively D =5.444, P<0.001 and D =5.167, P<0.001). CONCLUSIONS: Conservatively managed patients affected by TLJBF require a strict clinical follow-up since pain sometimes is present in the following months and it affects the quality of life. Surgery should be considered for these cases.

Surgery for vertebral involvement in multiple myeloma.

BACKGROUND: To evaluate the results of surgery for vertebral involvement in multiple myeloma (MM) following the introduction of the new chemotherapy in 2006. METHODS: Retrospective cohort study evaluating the patients with MM vertebral lesions from January 2006 to January 2014. Outcomes were based upon participation in ODI and EQ-5D. Statistical interpretation of the data was performed with SPSS version 19 software (SPSS Inc, Chicago, IL, USA). RESULTS: One hundred twenty patients affected by MM were potentially eligible for recruitment. About 1/3 patients needed an operation for vertebral involvement. The disability was mainly related to the number of vertebrae affected by MM. No difference in disability and quality of life was observed between operated and non-operated patients, according to age, levels, and follow-up. The operated patients with a Thoracic location reported lower disability and higher quality of life than non-operated patients. Non-operated patients with one or two vertebrae affected reported lower levels of quality of life. CONCLUSIONS: When a spinal column involvement occurs, the probability of undergoing surgery is about 40%. Operated patients show a moderate disability and the increased tendency is related to the number of vertebrae involved. The quality of life is middle in average and middle-high in patients operated for thoracic lesions. Nowadays the spinal surgeon must have a perspective view about MM vertebral lesions, evaluating the stability of the spine, the presence of deformity, the risk of vertebral fractures and the presence of drug resistant pain, that deteriorate the disability and the quality of life related to the underlying disease.

Lumbar ganglion cyst: Nosology, surgical management and proposal of a new classification based on 34 personal cases and literature review.

AIM: To analyze different terms used in literature to identify lumbar extradural cysts and propose a common scientific terminology; to elaborate a new morphological classification of this pathology, useful for clinical and surgical purposes; and to describe the best surgical approach to remove these cysts, in order to avoid iatrogenic instability or treat the pre-existing one. METHODS: We retrospectively reviewed 34 patients with symptomatic lumbar ganglion cysts treated with spinal canal decompression with or without spinal fixation. Microsurgical approach was the main procedure and spinal instrumentation was required only in case of evident pre-operative segmental instability. RESULTS: The complete cystectomy with histological examination was performed in all cases. All patients presented an improvement of clinical conditions, evaluated by Visual Analogic Scale and Japanese Orthopaedic Association scoring. CONCLUSION: Spinal ganglion cysts are generally found in the lumbar spine. The treatment of choice is the microsurgical cystectomy, which generally does not require stabilization. The need for fusion must be carefully evaluated: Pre-operative spondylolisthesis or a wide joint resection, during the operation, are the main indications for spinal instrumentation. We propose the terms "ganglion cyst" to finally identify this spinal pathology and for the first time its morphological classification, clinically useful for all specialists.

Static and dynamic cervical MRI: two useful exams in cervical myelopathy.

BACKGROUND: Cervical magnetic resonance imaging (MRI) is the gold standard exam in the assessment of patients affected by cervical myelopathy and is very useful in planning the operation. Herein we present a series of patients affected by long tract symptoms who underwent dynamic MRI in addition to the static exam. METHODS: In the period between March 2010 and March 2012, three-hundred-ten patients referred to our department since affected by neck/arm pain or symptoms related to cervical myelopathy. Thirty-eight patients complained "long-tract symptoms" related to cervical myelopathy. This series of patients was enrolled in the study. All patients underwent clinical and neurological exam. In all the cases, a static and dynamic cervical MRI was executed using a 3.0-T superconducting MR unit (Intera, Philips, Eindhoven, Netherlands). The dynamic exam was performed with as much neck flexion and extension the patient could achieve alone. On T2-weigthed MRI each level was assessed independently by two neuroradiologists and Muhle scale was applied. RESULTS: According to Muhle's classification of spinal cord compressions, static MRI demonstrated 156 findings: 96 (61.54%) anterior and 60 (38.46%) posterior. Dynamic MRI showed 186 spinal cord compressions: 81 (43.5%) anterior and 105 (56.5%) posterior. The anterior compressions were: grade 1 in 23 cases (28.4%), grade 2 in 52 cases (64.2%), grade 3 in 6 cases (7.4%). The posterior compressions were: 32 (30.48%) of grade 1, 60 (57.14%) of grade 2, 13 (12.38%) of grade 3. CONCLUSIONS: The dynamic MRI demonstrated a major number of findings and spinal cord compressions compared to the static exam. Finally, we consider the dynamic exam able to provide useful information in these patients, but we suggest a careful evaluation of the findings in the extension exam since they are probably over-expressed.

C5 palsy after insertion of a winged expandable cervical cage: a case report and literature review.

C5 nerve root palsy is a well-known complication after anterior or posterior cervical decompression. Many theories have been proposed but the etiology is still unclear. The use of a winged expandable cage after single or multiple corpectomy is among the used techniques in reconstructing the cervical spine. Herein we report a case of C5 palsy after a three-level corpectomy and reconstruction using this device for the treatment of cervical spondylosis. In our case the preexisting foraminal stenosis, wide anterior decompression and partial improvement of cervical alignment were factors supposed contributing to the palsy.

A case of cervical tuberculosis with severe kyphosis treated with a winged expandable cage after double corpectomy.

In this case report, we demonstrate that the use of a winged expandable cage was able to obtain good clinical and radiological results in a case of cervical tuberculosis with severe kyphosis. However, case series will be necessary to affirm its validity as a stand-alone device for similar cases with high risk of instability.

Revision surgery for degenerative spinal deformity: a case report and review of the literature.

INTRODUCTION: Management of spinal degenerative deformities always represents a challenge for the spinal surgeon. CASE PRESENTATION: We report a case of revision surgery for adult scoliosis, focusing of most common errors in pre-surgical management and criteria for reoperation. We analyzed the spino-pelvic parameters on the standing whole-spine X-ray and the role of sagittal balance. To restore 45° of lumbar lordosis, we performed a L3 Pedicle Subtraction Osteotomy (PSO), along with L2-L3 and L3-L4 eXtreme Lateral Interbody Fusion (XLIF). DISCUSSION: In cases of adult scoliosis, careful preoperative planning is necessary in an attempt to avoid difficult, expensive, and high-risk additional procedures.

Cervical reconstruction techniques. After adequate selection of the patient report of a series of 34 patients treated with winged expandable cages.

After single or multilevel cervical corpectomy for the treatment of different diseases, many surgical options have been proposed over the years. As a single device, a winged expandable titanium cage (WETC) has been developed. We report the use of this device and our clinical results at 24 months of follow-up in a prospective series of 34 patients who underwent single or multilevel corpectomy for the treatment of cervical spondylosis. Thirty-four consecutive patients underwent single or multilevel cervical corpectomy followed by the insertion of a WETC for the treatment of cervical spondylosis at Department of Neurology and Psychiatry "Sapienza" University of Rome, Italy, from May 2011 to May 2013. Patients affected by cervical spondylosis with cervical lordosis >0° were included. Patients affected by cervical lordosis <0°, osteoporosis, or other cervical diseases were excluded. The mean postsurgical Nurick's grade was significantly lower than the mean presurgical Nurick's grade (mean = 0.98 for postsurgical Nurick's grade and mean = 2.42 for presurgical Nurick's grade). Four patients (11.7 %) presented subsidence respectively of 1, 2, 2, and 3 mm at 24 months of radiological follow-up. Postoperative complications occurred in five patients (14.7 %). Our case series at 24 months of follow-up demonstrates that the use of a WETC obtains good clinical results after single or multiple corpectomy in patients not affected by osteoporosis who do not require cervical lordosis correction. During the insertion of the device, an adequate preparation of the end plates is suggested and their over distraction should be avoided.

Decompression versus decompression and fusion for degenerative lumbar stenosis: analysis of the factors influencing the outcome of back pain and disability.

BACKGROUND: The objective of this study is to evaluate the factors influencing the outcome of back pain and disability in patients operated for lumbar stenosis without instability and deformity using two classical surgical techniques: decompression alone and decompression plus fusion. METHODS: This is a retrospective cohort study of patients who underwent lumbar surgery with standard posterior decompression or standard posterior decompression plus pedicle screw fixation for degenerative lumbar stenosis without deformity, spondylolisthesis or instability at our department from June 2010 to January 2014. They were divided into two groups: decompression group (D) and decompression-fusion group (F). We analyzed the following factors: age, gender, levels of stenosis, pre-surgical "micro-instability", and post-surgical "micro-instability". RESULTS: A total of 174 patients were enrolled in the study. Both Graphic Rating Scale (GRS) and Oswestry Disability Index (ODI) scores were significantly decreased after surgery (P<0.001). Female patients appeared to have lesser improvements from surgery, for both D and F groups. An analysis of variance using the decrease of pain (GRS pre-post) as dependent variable and type of surgery, age, gender and their interaction as factors showed that the main effects of type of surgery and gender were significant. The analysis of variance for the decrease of pain (GRS) and disability (ODI) according to the levels of stenosis showed a significant interaction for GRS scores. Female patients that underwent fixation surgery reported the least improvement in disability. A significant interaction was found on the one-way analysis of variance for the D group without pre-surgical micro-instability using post-surgical micro-instability as factor. CONCLUSIONS: Our study supports posterior decompression alone as the gold standard option as treatment for lumbar stenosis without instability and deformity. Additional fusion should be considered only to prevent post-surgical instability. The "micro-instability" is a radiological finding that has its clinical surrogate but is not able to guide the choice of the type of surgery. Moreover the significance of "micro-instability" is still unclear. We suggest a prospective study following patients with asymptomatic micro-instability to definitively understand the clinical history.

Thoracic Spondylodiscitis Caused by Methicillin-resistant Staphylococcus aureus as a Superinfection of Pulmonary Tuberculous Granuloma in an Immunocompetent Patient: A Case Report.

Study Design Case report. Objective To describe a very rare case of an immunocompetent man who underwent surgery for thoracic spondylodiscitis caused by methicillin-resistant Staphylococcus aureus (MRSA) that developed as a superinfection of a pulmonary tuberculous granuloma. Methods Posterior decompression and pedicle screw vertebral fixation were followed by T5-T6 anterior somatotomy with implant of an expandable mesh and lateral plating as symptoms worsened. During the anterior approach, an atypical resection of the left lower lobe was also performed. Results A tuberculous granuloma was detected on histology. Ziehl-Neelsen stain confirmed the diagnosis. Culture also detected MRSA. Conclusions Early medical management is the first choice for spondylodiscitis to eradicate the infection and alleviate pain. Immobilization of the affected spine segments can protect the patient from vertebral collapse and from the appearance of neurologic deficits. Surgery is suggested if there are compressive effects on the spinal cord, spinal epidural abscess, vertebral collapse, and deformity. We decided to remove the abscess and to restore the anterior column using an anterior approach. Moreover, in this case, an anterior approach allowed us to identify the etiology of the lesion and to determine the best chemotherapy regimen.

Surgery in extensive vertebral hemangioma: case report, literature review and a new algorithm proposal.

Hemangiomas are benign dysplasias or vascular tumors consisting of vascular spaces lined with endothelium. Nowadays, radiotherapy for vertebral hemangiomas (VHs) is widely accepted as primary treatment for painful lesions. Nevertheless, the role of surgery is still unclear. The purpose of this study is to propose a novel algorithm of treatment about VHs. This is a case report of an extensive VH and a review of the literature. A case of vertebral fracture during radiotherapy at a total dose of 30 Gy given in 10 fractions (treatment time 2 weeks) using a linear accelerator at 15 MV high-energy photons for extensive VH is reported. Using PubMed database, a review of the literature is done. The authors have no study funding sources. The authors have no conflicting financial interests. In the literature, good results in terms of pain and neurological deficits are reported. No cases of vertebral fractures are described. However, there is no consensus regarding the treatment for VHs. Radiotherapy is widely utilized in VHs determining pain. Surgery for VHs determining neurological deficit is also widely accepted. Perhaps, regarding the width of the lesion, no indications are given. We consider it important to make an evaluation before initiating the treatment for the risk of pathologic vertebral fracture, since in radiotherapy, there is no convention regarding structural changes determined in VHs. We propose a new algorithm of treatment. We recommend radiotherapy only for small lesions in which vertebral stability is not concerned. Kyphoplasty can be proposed for asymptomatic patients in which VHs are small and in patients affected by VHs determining pain without spinal canal invasion in which the VH is small. In patients affected by pain without spinal canal invasion but in which the VH is wide or presented with spinal canal invasion and in patients affected by neurological deficits, we propose surgery.

Posterolateral arthrodesis in lumbar spine surgery using autologous platelet-rich plasma and cancellous bone substitute: an osteoinductive and osteoconductive effect.

Study Design Prospective cohort study. Objectives To analyze the effectiveness and practicality of using cancellous bone substitute with platelet-rich plasma (PRP) in posterolateral arthrodesis. Methods Twenty consecutive patients underwent posterolateral arthrodesis with implantation of cancellous bone substitute soaked with PRP obtained directly in the operating theater on the right hemifield and cancellous bone substitute soaked with saline solution on the right. Results Computed tomography scans at 6 and 12 months after surgery were performed in all patients. Bone density was investigated by comparative analysis of region of interest. The data were analyzed with repeated-measures variance analyses with value of density after 6 months and value of density after 12 months, using age, levels of arthrodesis, and platelet count as covariates. The data demonstrated increased bone density using PRP and heterologous cancellous block resulting in an enhanced fusion rate during the first 6 months after surgery. Conclusions PRP used with cancellous bone substitute increases the rate of fusion and bone density joining osteoinductive and osteoconductive effect.