Image Correlation Between Digitally Reconstructed Radiographs, C-arm Fluoroscopic Radiographs, and X-ray: A Phantom Study.

OBJECTIVE: Digitally reconstructed radiographs (DRRs) are planar two-dimensional (2D) X-rays derived from a three-dimensional (3D) computed tomography (CT) dataset. DRRs allow the simulation of radiographs of all desired views and facilitate preoperative planning. However, orthopedic surgeons rely on C-arm fluoroscopic imaging during surgery to verify fracture reduction and implant placement. Pincushion distortion represents a technical limitation of fluoroscopic imaging, resulting in a greater distance between points at the periphery of the image compared to the center. This project, therefore, aimed to assess the image correlation between digitally reconstructed radiographs (DRRs) and fluoroscopic imaging (C-arm) using conventional radiographs (X-ray) as a control. METHODS: A 3D-printed cubic prototype and an anatomical humerus bone model were used. C-arm fluoroscopic radiographs and conventional X-ray images were taken in an anteroposterior (AP) view at 10-degree steps while rotating the objects from 0 to 90 degrees. CT scans were made and used to compute and export DRRs in AP view at 10-degree rotational steps from 0 to 90 degrees. The surface area (cm2) was measured and compared between the different modalities. For automated image analysis of the anatomical humerus model, matching (%) between modalities was calculated using the structural similarity index (SSIM). RESULTS: The overall regression was statistically significant in all models, with an R2 >0.99 when comparing all three imaging modalities of the prototype. Surface correlation in the anatomical humerus model was R2 0.99 between X-ray and C-arm and R2 0.95 between C-arm and X-ray to DRRs, respectively. The SSIM was highest for comparing DRR and C-arm images (0.84±0.01%). CONCLUSIONS: The study indicates a strong agreement between digitally reconstructed radiographs and X-ray/C-arm images. DRRs, therefore, represent a valuable tool for research and clinical application.

Functional and Radiologic Outcomes of Degenerative Versus Traumatic Full-Thickness Rotator Cuff Tears Involving the Supraspinatus Tendon.

BACKGROUND: Arthroscopic rotator cuff repair (ARCR) is among the most commonly performed orthopaedic procedures. Several factors-including age, sex, and tear severity-have been identified as predictors for outcome after repair. The influence of the tear etiology on functional and structural outcome remains controversial. PURPOSE: To investigate the influence of tear etiology (degenerative vs traumatic) on functional and structural outcomes in patients with supraspinatus tendon tears. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Patients undergoing ARCR from 19 centers were prospectively enrolled between June 2020 and November 2021. Full-thickness, nonmassive tears involving the supraspinatus tendon were included. Tears were classified as degenerative (chronic shoulder pain, no history of trauma) or traumatic (acute, traumatic onset, no previous shoulder pain). Range of motion, strength, the Subjective Shoulder Value, the Oxford Shoulder Score (OSS), and the Constant-Murley Score (CMS) were assessed before (baseline) and 6 and 12 months after ARCR. The Subjective Shoulder Value and the OSS were also determined at the 24-month follow-up. Repair integrity after 12 months was documented, as well as additional surgeries up to the 24-month follow-up. Tear groups were compared using mixed models adjusted for potential confounding effects. RESULTS: From a cohort of 973 consecutive patients, 421 patients (degenerative tear, n = 230; traumatic tear, n = 191) met the inclusion criteria. The traumatic tear group had lower mean baseline OSS and CMS scores but significantly greater score changes 12 months after ARCR (OSS, 18 [SD, 8]; CMS, 34 [SD,18] vs degenerative: OSS, 15 [SD, 8]; CMS, 22 [SD, 15]) (P < .001) and significantly higher 12-month overall scores (OSS, 44 [SD, 5]; CMS, 79 [SD, 9] vs degenerative: OSS, 42 [SD, 7]; CMS, 76 [SD, 12]) (P≤ .006). At the 24-month follow-up, neither the OSS (degenerative, 44 [SD, 6]; traumatic, 45 [SD, 6]; P = .346) nor the rates of repair failure (degenerative, 14 [6.1%]; traumatic 12 [6.3%]; P = .934) and additional surgeries (7 [3%]; 7 [3.7%]; P = .723) differed between groups. CONCLUSION: Patients with degenerative and traumatic full-thickness supraspinatus tendon tears who had ARCR show satisfactory short-term functional results. Although patients with traumatic tears have lower baseline functional scores, they rehabilitate over time and show comparable clinical results 1 year after ARCR. Similarly, degenerative and traumatic rotator cuff tears show comparable structural outcomes, which suggests that degenerated tendons retain healing potential.

Gadolinium-Based Contrast Agents and Free Gadolinium Inhibit Differentiation and Activity of Bone Cell Lineages.

OBJECTIVES: Administration of gadolinium-based contrast agents (GBCA) in magnetic resonance imaging results in the long-term retention of gadolinium (Gd) in tissues and organs, including the bone, and may affect their function and metabolism. This study aims to investigate the effects of Gd and GBCA on the proliferation/survival, differentiation, and function of bone cell lineages. MATERIALS AND METHODS: Primary murine osteoblasts (OB) and osteoclast progenitor cells (OPC) isolated from C57BL/6J mice were used to test the effects of Gd3+ (12.5-100 µM) and GBCA (100-2000 µM). Cultures were supplemented with the nonionic linear Gd-DTPA-BMA (gadodiamide), ionic linear Gd-DTPA (gadopentetic acid), and macrocyclic Gd-DOTA (gadoteric acid). Cell viability and differentiation were analyzed on days 4-6 of the culture. To assess the resorptive activity of osteoclasts, the cells were grown in OPC cultures and were seeded onto layers of amorphous calcium phosphate with incorporated Gd. RESULTS: Gd3+ did not affect OB viability, but differentiation was reduced dose-dependently up to 72.4% ± 6.2%-73.0% ± 13.2% (average ± SD) at 100 µM Gd3+ on days 4-6 of culture as compared with unexposed controls (P < 0.001). Exposure to GBCA had minor effects on OB viability with a dose-dependent reduction up to 23.3% ± 10.2% for Gd-DTPA-BMA at 2000 µM on day 5 (P < 0.001). In contrast, all 3 GBCA caused a dose-dependent reduction of differentiation up to 88.3% ± 5.2% for Gd-DTPA-BMA, 49.8% ± 16.0% for Gd-DTPA, and 23.1% ± 8.7% for Gd-DOTA at 2000 µM on day 5 (P < 0.001). In cultures of OPC, cell viability was not affected by Gd3+, whereas differentiation was decreased by 45.3% ± 9.8%-48.5% ± 15.8% at 100 µM Gd3+ on days 4-6 (P < 0.05). Exposure of OPC to GBCA resulted in a dose-dependent increase in cell viability of up to 34.1% ± 11.4% at 2000 µM on day 5 of culture (P < 0.001). However, differentiation of OPC cultures was reduced on day 5 by 24.2% ± 9.4% for Gd-DTPA-BMA, 47.1% ± 14.0% for Gd-DTPA, and 38.2% ± 10.0% for Gd-DOTA (P < 0.001). The dissolution of amorphous calcium phosphate by mature osteoclasts was reduced by 36.3% ± 5.3% upon incorporation of 4.3% Gd/Ca wt/wt (P < 0.001). CONCLUSIONS: Gadolinium and GBCA inhibit differentiation and activity of bone cell lineages in vitro. Thus, Gd retention in bone tissue could potentially impair the physiological regulation of bone turnover on a cellular level, leading to pathological changes in bone metabolism.

MRI findings of traumatic and degenerative rotator cuff tears and introduction of the "cobra sign".

Background: A rotator cuff tear (RCT) is a common shoulder diagnosis and its etiology may be acute, traumatic, or chronic degenerative. Differentiation between the 2 etiologies may be important for multiple reasons, but remains difficult based on imaging. Further knowledge about radiographic and magnetic resonance findings to distinguish traumatic from degenerative RCT is needed. Methods: We analyzed magnetic resonance arthrograms (MRAs) of 96 patients with traumatic or degenerative superior RCT, which were matched according their age and the affected rotator cuff muscle into the 2 groups. Patients older than 66 years of age were excluded from the study to avoid including cases with pre-existing degeneration. In the case of traumatic RCT, the time between the trauma and MRA had to be less than 3 months. Various parameters of the supraspinatus (SSP) muscle-tendon unit were assessed (tendon thickness, presence of a remaining tendon stump at the greater tubercle, magnitude of retraction, layer appearance). The retraction of the 2 SSP layers were individually measured to determine the difference of retraction. Additionally, edema of the tendon and muscle, the tangent- and kinking-sign as well as the newly introduced Cobra-sign (bulging of the distal part of the ruptured tendon with slim configuration of the medial part of the tendon) were analyzed. Results: Edema within the SSP muscle (sensitivity 13%, specificity 100%, P = .011) or the tendon (sensitivity 86%, specificity 36%, P = .014) are more frequent in traumatic RCT. The same association was found for the kinking-sign (sensitivity 53%, specificity 71%, P = .018) and the Cobra sign (sensitivity 47%, specificity 84%, P = .001). Even though not statistically significant, tendencies were observed toward thicker tendon stumps in traumatic RCT, and greater difference in retraction between the 2 SSP layers in the degenerative group. The cohorts had no difference in the presence of a tendon stump at the greater tuberosity. Conclusion: Muscle and tendon edema, as well as tendon kinking appearance and the newly introduced cobra-sign are suitable MRA parameters to distinguish between traumatic and degenerative etiology of a superior RTC.

Standardized multi-planar reformation improves the reliability of the assessment of the anterolateral ligament in ACL-deficient knees.

PURPOSE: The anterolateral ligament (ALL) is an important structure for controlling anterolateral rotatory stability of the knee. Its assessment, however, is difficult using standardized MRI images. The goal of this study was to assess the reliability of judging the integrity of the ALL on multi-planar reformatted (MPR) MRI images and on standard coronal reformatted (SCR) MRI images in knees with an anterior cruciate ligament (ACL) rupture. METHODS: Forty-eight patients (14 females, 34 males, 30 ± 6 years (mean age ± standard deviation)) with acute ACL ruptures (< 2 weeks) and no additional knee injuries (except segond fractures) were included. Images were assessed by two independent raters twice with at least a 2-week interval in between. The assessment was first performed on SCR images and thereafter on MPR images. Images were judged for assessability of the ALL and then the integrity of the ALL was rated. RESULTS: Depending on rater and read, the ALL was judged as "torn" in between 5 (10.4%) and 11 (22.9%) patients out of 48 patients on SCR images. On MRP images, the ALL was judged as "torn" in between 5 (10.4%) and 6 (12.5%) patients out of 48 patients, depending on rater and read. Inter- and intra-rater reliability for the assessment of the ALL using MPR images was "substantial" to "almost perfect". Inter- and intra-rater reliability for the assessment using SCR was "fair" to "substantial". CONCLUSION: MPR images should be used when assessing the integrity of the ALL. Assessment quality is independent of patient positioning during MRI acquisition and the ALL can be displayed in full length on one image. LEVEL OF EVIDENCE: Level III.

The robustness of glenohumeral centering measurements in dependence of shoulder rotation and their predictive value in shoulders with rotator cuff tears.

OBJECTIVE: De-centering of the shoulder joint on radiographs is used as indicator for severity of rotator cuff tears and as predictor for clinical outcome after surgery. The objective of the study was to assess the effect of malrotation on glenohumeral centering on radiographs and to identify the most reliable parameter for its quantification. SUBJECTS AND METHODS: In this retrospective study (2014-2018), 249 shoulders were included: 92 with imaging-confirmed supra- and infraspinatus tears (rupture; 65.2 ± 9.9 years) and 157 without tears (control; 41.1 ± 13.0 years). On radiographs in neutral position and external rotation, we assessed three radiographic parameters to quantify glenohumeral centering: acromiohumeral distance (ACHD), craniocaudal distance of the humeral head and glenoid center (Deutsch), and scapulohumeral arch congruity (Moloney). Non-parametric statistics was performed. RESULTS: In both positions, only the distance parameters ACHD (< 0.5 mm) and Deutsch (< 1 mm) were comparable in the two study groups rupture and control. Comparing the parameters between the study groups revealed only ACHD to be significantly different with a reduction of more than 2 mm in the rupture group. Among the parameters, ACHD ≤ 6 mm was the only cut-off discriminating rupture (12-21% of the shoulders with ACHD ≤ 6 mm) and control (none of the shoulders with ACHD ≤ 6 mm). Ninety percent of shoulders with ACHD ≤ 6 mm presented with a massive rotator cuff tear (defined as ≥ 67% of the greater tuberosity exposed). CONCLUSION: Glenohumeral centering assessed by ACHD and Deutsch is not affected by rotation in shoulders with and without rotator cuff tear. An ACHD ≤ 6 mm has a positive predictive value of 90% for a massive rotator cuff tear.

Treatment of Knee Dislocation With Primary Repair and Suture Augmentation: A Viable Solution.

Background: Different surgical techniques have been described for the treatment of knee dislocation (KD). Nonoperative approaches are frequently combined with surgical reconstruction using auto- or allograft. Purpose: To evaluate the midterm results of primary surgical repair and suture augmentation to treat KD. Study Design: Case series; Level of evidence, 4. Methods: A total of 22 patients (5 women, 17 men; mean age, 45 ± 15 years) with KD were evaluated at a mean of 49 ± 16 months after surgical treatment that included primary repair and suture augmentation. Magnetic resonance imaging, stress radiographs, and outcome scores were obtained at the follow-up. Clinical examination including hop tests and force measurements for flexion and extension was performed. Results: The mean difference in pre- to postinjury Tegner scores was -2 ± 1. The outcome scores showed mean values of 84 ± 15 (Lysholm), 73 ± 15 (International Knee Documentation Committee) and 65 ± 25 (Anterior Cruciate Ligament-Return to Sport after Injury scale). Compared with the uninjured knee, the range of motion of the injured knee was reduced by 21° ± 12°. Twelve patients felt fit enough to perform hop tests and showed a mean deficit of 7% ± 17%° compared with the uninjured leg. The mean force deficit was 19% ± 18% for extension and 8% ± 16% for flexion. Stress radiographs revealed an 11 ± 7-mm higher anteroposterior translation on the injured side. Four patients had secondary ligament reconstructions due to persistent instability and 7 underwent arthroscopic arthrolysis due to stiffness. A significant increase of osteoarthritis was found for the medial, lateral, and patellofemoral compartments (P = .007, .004, and .006, respectively). Conclusion: Primary repair and suture augmentation of KD led to satisfactory clinical midterm results despite persistent radiological instability and a significant increase in osteoarthritis. This technique allows the return to activities of daily living without subjective instability in most nonathletic patients. Secondary ligament reconstructions should be performed if relevant instability persists to decrease the risk of secondary meniscal and cartilage damage.

Multiplanar reformation improves identification of the anterolateral ligament with MRI of the knee.

The anterolateral ligament (ALL) is subject of the current debate concerning rotational stability in case of anterior cruciate ligament (ACL) injuries. Today, reliable anatomical and biomechanical evidence for its existence and course is available. Some radiologic studies claim to be able to identify the ALL on standard coronal plane MRI sections. In the experience of the authors, however, ALL identification on standard MRI sequences frequently fails and is prone to errors. The reason for this mainly lies in the fact, that the entire ALL often cannot be identified on a single MRI image. This study aimed to establish an MRI evaluation protocol improving the visualization of the ALL, using multiplanar reformation (MPR) with the goal to be able to evaluate the ALL on one MRI image. A total of 47 knee MRIs performed due to atraumatic knee pain between 2018 and 2019 without any pathology were analyzed. Identification of the ALL was performed twice by an orthopedic surgeon and a radiologist on standard coronal plane and after MPR. For the latter axial and coronal alignment was obtained with the femoral condyles as a reference. Then the coronal plane was adjusted to the course of the ALL with the lateral epicondyle as proximal reference. Visualization of the ALL was rated as "complete" (continuous ligamentous structure with a tibial and femoral insertion visible on one coronal image), "partial" (only parts of the ALL like the tibial insertion were visible) and "not visible". The distances of its tibial insertion to the bony joint line, Gerdy's tubercle and the tip of the fibular head were measured. On standard coronal images the ALL was fully visible in 17/47, partially visible in 27/47, and not visible in 3/47 cases. With MPR the ALL was fully visible in 44/47 and not visible in 3/47 cases. The median distance of its tibial insertion to the bony joint line, Gerdy's tubercle and the tip of the fibular head were 9, 21 and 25 mm, respectively. The inter- (ICC: 0.612; 0.645; 0.757) and intraobserver (ICC: 0.632; 0.823; 0.857) reliability was good to excellent. Complete visualization of the ALL on a single MRI image is critical for its identification and evaluation. Applying multiplanar reformation achieved reliable full-length visualization of the ALL in 94% of cases. The described MPR technique can be applied easily and fast in clinical routine. It is a reliable tool to improve the assessment of the ALL.

The EOS 3D imaging system reliably measures posterior tibial slope.

BACKGROUND: One of the values determined during the assessment of knee issues is the posterior tibial slope (PTS). A new option for measuring the PTS is the EOS 3D imaging system, which provides anteroposterior (AP) and lateral long leg radiographs (LLRs) using less radiation than a conventional LLR. We investigated the reliability of the EOS 3D imaging system with respect to PTS measurements. METHODS: We retrospectively searched our radiological database for patients who underwent an EOS scan and a computed tomography (CT) scan of their lower extremities between January and December 2019. Fifty-six knees were included in the study. Medial and lateral PTSs were determined using both modalities. A radiologist and an orthopaedic surgeon each performed all measurements twice and the intraclass correlation (ICC) was calculated to assess inter- and intrarater reliability. The Student t test and Pearson correlation were used to compare the results of both imaging modalities. RESULTS: The mean medial PTS was 8.5° (95% confidence interval [CI], 8.1-8.9°) for the EOS system and 7.7° (95% CI, 7.3-8.1°) for CT, and the lateral PTS was 7.4° (95% CI, 6.9-7.9°) for the EOS system, and 7.0° (95% CI, 6.5-7.4°) for CT. Interrater reliability (ICC) with respect to medial and lateral PTSs measured on the EOS (0.880, 0.765) and CT (0.884, 0.887) images was excellent. The intrarater reliability of reader 1 (ICC range, 0.889-0.986) and reader 2 (ICC range, 0.868-0.980) with respect to the same measurements was excellent. CONCLUSION: The PTS measurements from the EOS 3D imaging system are as reliable and reproducible as those from CT, the current gold standard method. We recommend using this system if possible, because it acquires more information (sagittal plane) in a scan than a conventional LLR, while exposing the patient to less radiation. LEVEL OF EVIDENCE: Level III, Retrospective cohort study.

Thermal treatment at 500 °C significantly reduces the reaction to irregular tricalcium phosphate granules as foreign bodies: An in vivo study.

Evaporation of phosphate species during thermal treatment (> 400 °C) of calcium phosphates leads to the formation of an alkaline layer on their surface. The aim of this study was to evaluate the hypothesis that the biological response of thermally treated calcium phosphates is modified by the presence of such an alkaline layer on their surface. For this purpose, 0.125-0.180 mm α- and ß-tricalcium phosphate (TCP) granules were obtained by crushing and size classification, with some being subjected to thermal treatment at 500 °C. The four types of granules (α-TCP, ß-TCP, α-TCP-500 °C, and ß-TCP-500 °C) were implanted subcutaneously and orthotopically in rats. Sham operations served as control. Subcutaneously, α-TCP and ß-TCP induced significantly more multinucleated giant cells (MNGCs) than calcined granules. Most of the induced MNGCs were TRAP-negative, CD-68 positive and cathepsin K-negative, reflecting a typical indication of a reaction with a foreign body. The vessel density was significantly higher in the α-TCP and ß-TCP groups than it was in the α-TCP-500 °C and ß-TCP-500 °C groups. In the femur model, ß-TCP-500 °C induced significantly more new bone formation than that induced by ß-TCP. The granule size was also significantly larger in the ß-TCP-500 °C group, making it more resistant to degradation than ß-TCP. The MNGC density was higher in the α-TCP and ß-TCP groups than in the α-TCP-500 °C and ß-TCP-500 °C groups, including cathepsin-positive, CD-68 positive, TRAP-positive and TRAP-negative MNGCs. In conclusion, this study confirms that the biological response of calcium phosphates was affected by the presence of an alkaline layer on their surface. Thermally-treated α-TCP and ß-TCP granules produced significantly fewer MNGCs and were significantly less degraded than non-thermally-treated α-TCP and ß-TCP granules. Thermally treating α-TCP and ß-TCP granules shifts the reaction from a foreign body reaction towards a physiological reaction by downregulating the number of induced MNGCs and enhancing degradation resistance.

A Novel Bioreactor System Capable of Simulating the In Vivo Conditions of Synovial Joints.

Any significant in vitro evaluation of cartilage tissue engineering and cartilage repair strategies has to be performed under the harsh conditions encountered in vivo within synovial joints. To this end, we have developed a novel automated physiological robot reactor system (PRRS) that is capable of recapitulating complex physiological motions and load patterns within an environment similar to that found in the human knee. The PRRS consists of a mechanical stimulation unit (MSU) and an automatic sample changer (ASC) within an environment control box in which the humidity, temperature, and gas composition are tightly regulated. The MSU has three linear (orthogonal) axes and one rotational degree of freedom (around the z-axis). The ASC provides space for up to 24 samples, which can be allocated to individual stimulation patterns. Cell-seeded scaffolds and ex vivo tissue culture systems were established to demonstrate the applicability of the PRRS to the investigation of the effect of load and environmental conditions on engineering and maintenance of articular cartilage in vitro. The bioreactor is a flexible system that has the potential to be applied for culturing connective tissues other than cartilage, such as bone and intervertebral disc tissue, even though the mechanical and environmental parameters are very different.

Bovine Osteochondral Tissues: A Questionable Model to Evaluate Mechanical Loading In Vitro.

Articular cartilage exists within synovial joints to adsorb and distribute mechanical loads to the subchondral bone. Mechanical loading is one aspect of a wide range of microenvironmental stressors that contribute to the maintenance of articular cartilage. The aim of the current study was to characterize bovine osteochondral tissues and to assess their suitability to serve as a model for investigating the effects of mechanical loading on cartilage tissue in vitro using a custom-made reactor system. Osteochondral tissues were harvested from bovine knee joints and cultured up to 24 days in loaded and unloaded conditions. Notably, we found a considerable zone-specific heterogeneity between cartilage explants harvested from the same joint as evidenced by histology and gene expression levels. Results using the reactor system revealed that differences observed after mechanical loading varied within the range of the heterogeneity observed amongst the different cartilage explants. Thus, it may be difficult to obtain reliable and reproducible data in mechanical loading experiments from these tissues in vitro, especially in cases where small variations between the experimental groups are expected. This will likely lead to the reporting of false positives or negatives in studies investigating the effect of mechanical load on the function of cartilage tissue.

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-ßs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-ß is essential for the formation of cartilage-like tissue and that alterations within the TGF-ß signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-ß1 was required to induce the formation of cartilage-like tissue. When TGF-ß signaling was blocked by inhibiting the TGF-ß receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-ß receptors 1 and 2 and TGF-ß2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-ß. We propose that a decrease in the expression of the chondrogenic growth factor TGF-ß2 and of the TGF-ß receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-ß signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.

Tissue engineering - nanomaterials in the musculoskeletal system.

The musculoskeletal tissues bone, cartilage and ligament/tendon are highly structured nanocomposites consisting of nanofibres embedded in a matrix of different composition. Thus, it was a logical step that during the hype of nano in the last decade, nanotechnology and nanomaterials became a hot topic in the field of musculoskeletal repair. Especially the fact that using nanomaterials would encompass a biomimetic approach, thus copying nature, was promising. However, it became evident that using nanomaterials in the repair of musculoskeletal tissues had a longer history than initially thought and its way was paved with failures, which are important to remember when applying current ideas. This current opinion paper summarises some fundamental aspects of nanomaterials to be used for musculoskeletal application and discusses where this field might move to in the near future.

Physiological cartilage tissue engineering effect of oxygen and biomechanics.

In vitro engineering of cartilaginous tissues has been studied for many years, and tissue-engineered constructs are sought to be used clinically for treating articular cartilage defects. Even though there is a plethora of studies and data available, no breakthroughs have been achieved yet that allow for implanting in vivo cultured articular cartilaginous tissues in patients. A review of contributions to cartilage tissue engineering over the past decades emphasizes that most of the studies were performed under environmental conditions neglecting the physiological situation. This is specifically pronounced in the use of bioreactor systems which neither allow for application of near physiomechanical stimulations nor for controlling a hypoxic environment as it is experienced in synovial joints. It is suspected that the negligence of these important parameters has slowed down progress and prevented major breakthroughs in the field. This review focuses on the main aspects of cartilage tissue engineering with emphasis on the relation and understanding of employing physiological conditions.

Chondrocytes within osteochondral grafts are more resistant than osteoblasts to tissue culture at 37°C.

It is proposed that an ideal osteochondral allograft for cartilage repair consists of a devitalized bone but functional cartilage. The different modes of nutrient supply in vivo for bone (vascular support) and cartilage (diffusion) suggest that a modulation of storage conditions could differentially affect the respective cells, resulting in the proposed allograft. For this purpose, osteochondral tissues from porcine humeral heads were either cultured at 37°C for up to 24 hr or stored at 4°C for 24 hr, the temperature at which osteochondral allografts are routinely stored. Functionality of the cells was assessed by in situ hybridization for transcripts encoding collagen types I and II. At 37°C, a time-dependent significant reduction of the bone surface covered with functional cells was observed with only 5% ± 5% coverage left at 24 hr compared with 41% ± 10% at 0 hr. Similarly, cartilage area containing functional cells was significantly reduced from 84% ± 7% at 0 hr to 70% ± 3% after 24 hr. After 24 hr at 4°C, a significantly reduced amount of functional cells covering bone surfaces was observed (27% ± 5%) but not of cells within the cartilage (79% ± 8%). In the applied experimental setup, bone cells were more affected by tissue culture at 37°C than cartilage cells. Even though chondrocytes appear to be more sensitive to 37°C than to 4°C, the substantially reduced amount of functional bone cells at 37°C warrants further investigation of whether a preincubation of osteochondral allografts at 37°C--prior to regular storage at 4°C--might result in an optimized osteochondral allograft with devitalized bone but viable cartilage.

Chondrocyte function after osteochondral transfer: comparison of concave and plane punches.

BACKGROUND: An incongruity between instrument and articular surfaces in osteochondral transfer (OCT) results in unevenly distributed impact forces exerted on the cartilage which may cause a loss of functional chondrocytes. We tested whether a plane instead of a concave design of the punch of an osteotome can reduce these cartilage damages. METHODS: Osteochondral cylinders were transferred from a donor to a recipient site within porcine humeral heads. Histological sections of the cartilage were assessed for metabolic active chondrocytes by in situ hybridization detecting coll alpha(1)(II) mRNA subsequent to OCT and 24 h thereafter. RESULTS: The percentage of cartilage harbouring functional chondrocytes in the transferred grafts was 85 +/- 10 and 91 +/- 4% subsequently to OCT using punches with concave or plane surfaces, respectively, and 83 +/- 10% (concave) and 82 +/- 10% (plane) after 24 h. In the superficial layer of the cartilage the percentages were 72 +/- 13% (concave) and 84 +/- 8% (plane) subsequently to OCT, and 68 +/- 15% (concave) and 70 +/- 3% (plane) after 24 h. The analysis did not reveal any statistically significant differences. CONCLUSIONS: The OCT leads to considerable loss of functional chondrocytes which could not be prevented by the use of a plane instead of a concave punch. Since functional chondrocytes might be of crucial importance for the survival and integration of the graft into the recipient site further work is needed to optimize the OCT procedure.

Functional analysis of the osteoarthritis susceptibility-associated GDF5 regulatory polymorphism.

OBJECTIVE: Single-nucleotide polymorphism (SNP) rs143383 (T to C) in the 5'-untranslated region (5'-UTR) of GDF5 has recently been reported to be associated with osteoarthritis (OA) susceptibility, with lower expression of the risk-associated T allele observed in vitro and in vivo. The in vivo studies were performed on cartilage tissue from OA patients. The present study was undertaken to expand the analysis of the effect of this SNP on GDF5 allelic expression to more joint tissue types, to investigate for cis and trans factors that interact with the SNP, and to examine novel cis-acting GDF5 regulatory polymorphisms. METHODS: Tissue samples were collected from OA patients undergoing joint replacement of the hip or knee. Nucleic acid was extracted, and, using rs143383 and an assay that discriminates and quantifies allelic expression, the relative amount of GDF5 expression from the T and C alleles was measured. Additional common variants in the GDF5 transcript sequence were interrogated as potential regulatory elements using allelic expression and luciferase reporter assays, and electrophoretic mobility shift assays were used to search for trans factors binding to rs143383. RESULTS: We observed a consistent allelic expression imbalance of GDF5 in all tissues tested, implying that the functional effect mediated by rs143383 on GDF5 expression is joint-wide. We identified a second polymorphism, located in the 3'-UTR of GDF5, that influenced allelic expression of the gene independent of rs143383. Finally, we observed differential binding of deformed epidermal autoregulatory factor 1 (DEAF-1) to the 2 alleles of rs143383. CONCLUSION: These findings show that the OA susceptibility mediated by polymorphism in GDF5 is not restricted to cartilage, emphasizing the need to consider the disease as involving the whole joint. The existence of an additional cis-acting regulatory polymorphism highlights the complexity of the regulation of expression of this important OA susceptibility locus. DEAF-1 is a trans-acting factor that merits further investigation as a potential tool for modulating GDF5 expression.

Time course of biological activity in fresh murine osteochondral allografts paralleled to the recipient's immune response.

The use of fresh osteochondral allografts is a popular approach to treat articular cartilage lesions. Immunological reactions of the recipient elicited by the allograft's osseous portion, however, frequently result in their deterioration. So far, little emphasis has been put on describing morphology and biological activity in fresh allografts and paralleling these to the immunological processes triggered in the host. Therefore, in the present study murine neonatal femora, serving as osteochondral grafts, were transplanted as fresh isografts (controls) or allografts (the latter in non- or presensitized mice) and retrieved after 2, 5, 10, and 20 days. It was shown that (1) in isografts active bone cells (osteoblasts, osteoclasts) were present, the bone marrow was repopulated with hematopoietic cells, the diaphysis increased in length, and no specific immunological reaction by the recipient was evoked. (2) Allografts transplanted into nonsensitized hosts initially appeared similar as isografts, but activated T lymphocytes at the transplantation site preceded loss of active bone cells within the graft and development of fibrosis within the marrow cavity. (3) In allografts transplanted into presensitized recipients, severe deterioration of the graft was observed with very few active bone cells, accompanied by an invasion of T lymphocytes and fibrosis in the marrow cavity already in early stages. Similar to vital organ transplantation, the function of cells within osteochondral allografts is severely impaired after being recognized by the immune system. Therefore, emphasis has to be placed on the development of procedures preserving cartilage biology while reducing the antigenicity of the allograft's osseous portion.