Repeated intra-articular injections of umbilical cord-derived mesenchymal stem cells for knee osteoarthritis: a phase I, single-arm study.

INTRODUCTION: Stem cell therapy has emerged as an effective treatment for multiple diseases, and some studies also demonstrate that it may be a promising treatment for osteoarthritis (OA). However, few studies have clarified the safety of repeated intra-articular injection of human umbilical cord-derived mesenchymal stem cells (UC-MSCs). To promote its application in treating OA, we conducted an open-label trial to investigate the safety of repeated intra-articular injections of UC-MSCs. METHODS: Fourteen patients with OA (Kellgrene-Lawrence grade 2 or 3) who received repeated intra-articular injections of UC-MSCs were evaluated in three months of follow-up. The primary outcomes were the adverse events, and the second outcomes included visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scores and SF-12 quality of life score. RESULTS: A total of 5 of 14 patients (35.7%) experienced transient adverse reactions, which resolved spontaneously. All patients showed some improvement in knee function limitation and pain after receiving stem cell therapy. VAS score 6.0 to 3.5, WOMAC score 26.0 to 8.5, MOCART score 42.0 to 58.0, SF-12 score 39.0 to 46.0. CONCLUSION: Repeated intra-articular injection of UC-MSCs demonstrates safety in treating OA and does not induce serious adverse events. This treatment may transiently improve symptoms in patients with knee OA and may be a potential therapeutic option for OA.

Tissue Regrowth and Its Vascularization Through Bone Marrow Stimulation: Microfracture at the Acetabular Rim for Irreparable Labral Tear in a Porcine Model.

BACKGROUND: The treatment of irreparable acetabular labral tear remains a great challenge. Whether fibrocartilage-like tissue can regrow with sufficient volume to fill the labral defect area through bone marrow stimulation remains unknown. PURPOSE: To characterize the healing process and vascularization course of the regrown tissue after microfracture at the acetabular rim for irreparable labral tears in a porcine model. STUDY DESIGN: Descriptive laboratory study. METHODS: Twelve pigs randomly underwent unilateral microfracture at the acetabular rim after the resection of a 10 mm-long section of labrum from 10 to 1 o'clock. Pigs were randomly sacrificed at 6 and 12 weeks postoperatively. The regrown tissues were harvested for macroscopic evaluation and histologic assessment. The regrown tissue was zoned into 2 halves to observe the vascular distribution: the capsular half (zone I) and the articular half (zone II). Each zone was divided into 2 parts: the peripheral part (IA and IIA) and the part attached to the acetabulum (IB and IIB). RESULTS: At 6 weeks, all regrown tissue was hypotrophic with <50% filling of the labral defect. Fibrochondrocytes were concentrated at the interface between the acetabulum and the regrown tissue. The vascularization was equal among each part within the regrown tissue. As compared with regrown tissue at 12 weeks, proteoglycan and collagen type 1 and 2 were more evident within the regrown tissue at 6 weeks. At 12 weeks, tissue disintegration occurred in all regrown tissue with <25% filling of the labral defect area. The vascular structure could barely be observed, with few fibrochondrocytes found at the area adjacent to the acetabulum. CONCLUSION: Fibrocartilage-like tissue did regrow with well-distributed vascular ingrowth of each part of the regrown tissue through bone marrow stimulation at the early stage. However, insufficient volume of the regrown tissue led to loss of the hip suction seal and subsequent tissue disintegration. CLINICAL RELEVANCE: Microfracture at the rim of the acetabulum alone could not restore the morphology and function of the acetabular labrum. Nonetheless, microfracture at the acetabular rim might be a viable adjunct to labral reconstruction, as the well-distributed vascularization through bone marrow stimulation might overcome the obstacle of poor vascular ingrowth of the articular half of the autograft.

Tissue Integration and Chondroprotective Potential of Acetabular Labral Augmentation with Autograft Tendon: Study of a Porcine Model.

BACKGROUND: The aim of this study was to explore the tissue healing process and changes in articular cartilage following acetabular labral augmentation in a porcine model. METHODS: The labrum was resected unilaterally from 10 o'clock to 1 o'clock on the capsular side in 36 pigs. Eighteen pigs underwent labral augmentation (AUG group) using autologous Achilles tendon. No additional procedures were performed in the remaining pigs (control group). The pigs were killed at 6, 12, or 24 weeks postoperatively for histological assessment and measurement of the inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α in synovial fluid. RESULTS: All autografts were well placed in the labral defect in the AUG group, and good integration of the autograft with the remnant chondrolabral junction was observed at 24 weeks; only scar tissue was observed in the control group at 6, 12, and 24 weeks. Fibrochondrocytes were concentrated at the transition between the autograft and native labrum at early time points, and the cells within the autograft labrum were predominantly fibrochondrocytes at 24 weeks. Rough and irregular articular cartilage surfaces were observed in 3 of the 6 samples in the AUG group at 24 weeks; the others appeared smooth. Focal cartilage erosion (predominantly in the acetabulum) occurred in all samples in the control group at 12 and 24 weeks. The Mankin score at 24 weeks was significantly lower in the AUG group than in the control group (mean [95% confidence interval]: 2.33 [1.06 to 3.6] versus 9 [8.06 to 9.94], p < 0.001). Likewise, the concentrations of all cytokines (in pg/mL) were significantly lower in the AUG group than in the control group at 24 weeks (IL-6: 166.6 [155.22 to 177.94] versus 245.9 [242.66 to 249.14], p < 0.001; IL-1ß: 122.1 [116.4 to 127.83] versus 282.9 [280.29 to 285.51], p < 0.001; and TNF-α: 56.22 [53.15 to 59.29] versus 135 [131.66 to 138.24], p < 0.001). CONCLUSIONS: Autograft tendon used for labral augmentation was able to integrate well with the native labrum, which may help to preserve the articular cartilage. CLINICAL RELEVANCE: Labral augmentation with autograft tendon may be a feasible option in cases of viable labral remnants.

Application of three-dimensional-printed porous tantalum cones in total knee arthroplasty revision to reconstruct bone defects.

Purpose: Three-dimensional (3D) printing technology has emerged as a new treatment method due to its precision and personalization. This study aims to explore the application of a 3D-printed personalized porous tantalum cone for reconstructing the bone defect in total knee arthroplasty (TKA) revision. Methods: Between November 2017 and October 2020, six patients underwent bone reconstruction using 3D-printed porous tantalum cones in TKA revision. The knee function was assessed using the Hospital for Special Surgery (HSS) score pre- and postoperatively. The pain was measured by the visual analog scale (VAS) pre- and postoperatively. The quality of life was measured using the 36-Item Short Form Health Survey (SF-36) to pre- and postoperatively evaluate the relief of pain. Operation time, intraoperative blood loss, postoperative drainage volume, and complications were also recorded. At the last follow-up, all patients received X-ray and computed tomography (CT) to confirm the effect of bone reconstruction. Results: After an average follow-up duration of 26.3 months, no patients developed any operation-related complications. The average intraoperative blood loss and postoperative drainage volumes were 250.1 ± 76.4 ml and 506.7 ± 300.8 ml, respectively. At the last follow-up, the HSS score was significantly higher than that before operation, indicating that the knee function was significantly improved (p < 0.001). During the follow-up, the mean VAS score decreased and the mean SF-36 score increased, both of which were significantly improved compared with preoperative conditions (p < 0.001). Radiological examination at the final follow-up showed that cones implanted into the joint were stable and bone defects were effectively reconstructed. Conclusion: This study demonstrated that 3D-printed porous tantalum cones could effectively reconstruct bone defects and offer anatomical support in TKA revision. Further studies are still needed to confirm the long-term effect of 3D-printed tantalum cones for reconstructing bone defects.

The Vascularization Course of Labral Autograft and Its Effect on Tissue Healing: Acetabular Labral Augmentation Versus Labral Reconstruction in a Porcine Model.

BACKGROUND: The preservation of the native labral vascularization is assumed to be the potential advantage of acetabular labral augmentation, the effect of which remains unknown. PURPOSE: To identify the vascular distribution within the labral autograft and its effect on the healing process between labral augmentation (AUG) and reconstruction (RECON) in a porcine model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 36 pigs randomly underwent unilateral labral augmentation or reconstruction (AUG group, n = 18; RECON group, n = 18). The pigs were randomly sacrificed at 6, 12, and 24 weeks postoperatively. The labral autografts were harvested for macroscopic evaluation and histologic assessment. The labral autograft was zoned into 2 halves to observe the vascular distribution: the capsular half (zone I) and the articular half (zone II). Each zone was divided into 2 parts: the peripheral part (IA and IIA) and the part attached to the acetabulum (IB and IIB). RESULTS: At 6 weeks, there existed more vascular ingrowth in zone I, whereas zone IIB appeared nearly avascular in both groups. At 12 weeks, the area with the greatest vascularity was zone II in the RECON group and zone IA in the AUG group. The vascularity was concentrated at zones IA and IIA in both groups at 24 weeks. The labral autografts were hypertrophic with sufficient filling of the labral defect in both groups at 6 weeks. At 12 weeks, an insufficient volume of the articular half was observed in 3 of 6 labral autografts in the RECON group, while all autografts remained well integrated with the chondrolabral junction in the AUG group. At 24 weeks, unsatisfactory merging of the labral autograft with the cartilage at the articular side was found in 2 of 6 labral autografts in the RECON group, which was not observed in the AUG group despite the sufficient volume of autografts labrum in both groups. CONCLUSION: Slow vascular ingrowth within the articular half might account for the poor healing of the reconstructed labral autograft. Labral augmentation provides the possibility of better tissue healing because of the preservation of the original chondrolabral junction compared with labral reconstruction. CLINICAL RELEVANCE: Labral augmentation might be a feasible alternative to labral reconstruction under the condition of viable labral remnants.

Advanced Hydrogels With Nanoparticle Inclusion for Cartilage Tissue Engineering.

Cartilage dysfunctions caused by congenital disease, trauma and osteoarthritis are still a serious threat to joint activity and quality of life, potentially leading to disability. The relatively well-established tissue engineering technology based on hydrogel is a promising strategy for cartilage defect repairing. However, several unmet challenges remain to be resolved before its wide application and clinical translation, such as weak mechanical property and compromised bioactivity. The development of nanomedicine has brought a new dawn to cartilage tissue engineering, and composite hydrogel containing nanoparticles can substantially mimic natural cartilage components with good histocompatibility, demonstrating unique biological effects. In this review, we summarize the different advanced nanoparticle hydrogels currently adopted in cartilage tissue engineering. In addition, we also discuss the various application scenarios including injection and fabrication strategies of nanocomposite hydrogel in the field of cartilage repair. Finally, the future application prospects and challenges of nanocomposite hydrogel are also highlighted.

Hydrogel composed of type II collagen, chondroitin sulfate and hyaluronic acid for cartilage tissue engineering.

BACKGROUND: Cartilage tissue engineering is a promising way to repair cartilage defects. Different materials have been applied in the preparation of cartilage hydrogels, but all with various disadvantages. OBJECTIVE: The aim of this study was to prepare cartilage hydrogel using type II collagen, chondroitin sulfate and hyaluronic acid, to explore their gelation effect and compressive strength, and to analyze the feasibility of their application in cartilage tissue engineering. METHODS: Type II collagen (Col II), hyaluronic acid (HA) and chondroitin sulfate (CS) were mixed in a certain proportion to prepare gel scaffolds; changes in chemical groups were detected by Fourier transform infrared. After the hydrogel was prepared, its compressive strength was measured. Umbilical cord stem cells were co-cultured with hydrogel scaffolds to observe its cytocompatibility and analyze whether stem cells had cellular activity during co-culture; histological staining was applied to observe the hydrogel loaded with stem cells. RESULTS: Cartilage hydrogels were successfully prepared with good compressive strength, and Fourier transform infrared analysis showed that Schiff base reaction occurred during the preparation process and tight chemical cross-linking was formed. The results of umbilical cord stem cell co-culture showed that the hydrogel had good cytocompatibility and the stem cells had good activity in the hydrogel. CONCLUSIONS: Cartilage hydrogels with stable structures were successfully prepared and had good compressive strength. Hydrogel scaffold could provide a suitable living environment for umbilical cord stem cells, so that they maintain normal cell morphology and activity, and has a good application potential in cartilage tissue engineering.

Bilateral pulmonary embolism without deep venous thrombosis was observed after knee arthroscopy: a case report.

BACKGROUND: Symptomatic pulmonary embolism (PE) after knee arthroscopy is extremely rare. If the embolism is not treated promptly, the patient may die. Bilateral pulmonary embolism with associated pulmonary infarct without concomitant deep vein thrombosis has never been reported following routine knee arthroscopy. CASE PRESENTATION: A 50-year-old female patient with no other risk factors other than hypertension, obesity, varicose veins in the ipsilateral lower extremities and elevated triglyceride (TG) presented to our ward. She had experienced sudden chest tightness, polypnea and fainting after going to the bathroom the morning of the second postoperative day and received emergency medical attention. Colour ultrasonography of the extremities showed no deep vein thrombosis. Lung computed tomography angiography (CTA) showed multiple embolisms scattered in both pulmonary artery branches. Thus, emergency interventional thrombolysis therapy was performed, followed by postoperative symptomatic treatment with drugs with thrombolytic, anticoagulant and protective activities. One week later, lung CTA showed a significant improvement in the PEs compared with those in the previous examination. Since the aetiology of PE and no obvious symptoms were discerned, the patient was discharged. CONCLUSION: Although knee arthroscopy is a minimally invasive and quick procedure, the risk factors for PE in the perioperative period should be considered and fully evaluated to enhance PE detection. Moreover, a timely diagnosis and effective treatment are important measures to prevent and cure PE after knee arthroscopy. Finally, clear guidelines regarding VTE thromboprophylaxis following knee arthroscopy in patients with a low risk of VTE development are needed.

Proliferation ability of particulated juvenile allograft cartilage.

BACKGROUND: Particulated juvenile allograft cartilage (PJAC) has a good short-term clinical efficacy in repairing articular cartilage defects, but the proliferation ability of PJAC and the biological characteristics of transplanted cells after transplantation are still unclear. PURPOSE: To study the cartilage proliferation ability of PJAC in repairing full-thickness cartilage defects and the reasons for proliferation to provide experimental evidence for its clinical application. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty Guizhou minipigs were randomly divided into the experimental group and control group. In all minipigs, an 8-mm cylindrical full-thickness cartilage defect was created in the femoral trochlea of one knee. The experimental group received PJAC transplantation from five juvenile donors of Guizhou minipigs (PJAC group; n = 10) and the control group received transplantation of autologous cartilage chips (ACC group; n = 10). Both groups were followed at 1 and 3 months after surgery, immunohistochemical evaluation of the tissue sections Ki-67 and Lin28 was conducted, the positive rate was calculated according to the staining, and the proliferation ability of PJAC was analyzed. RESULTS: All 20 Guizhou minipigs were followed, and there was no infection or incision healing disorder after surgery. By Ki-67 and Lin28 immunohistochemical tests, the positive rate of Ki-67 was 88.9 ± 0.2% in the PJAC group and 28.3 ± 3.6% in the ACC group at 1 month, and the difference was statistically significant (P < 0.05); the positive rate of Lin28 was 34.6 ± 3.3% in the PJAC group and 7.6 ± 1.4% in the ACC group at 1 month, and the difference was statistically significant (P < 0.05). At 3 months, the positive rates of Ki-67 in the PJAC group and ACC group were 53.6 ± 6.9% and 1.97 ± 0.3%, respectively (P < 0.05); the positive rates of Lin28 were 86.6 ± 3.3% and 1.4 ± 0.3%, respectively (P < 0.01). CONCLUSION: A large animal model was established with Guizhou minipigs, and the expressions of Ki-67 protein and Lin28 protein detected by immunohistochemistry in the repaired transplanted tissue of the PJAC group were stronger than those of adult cartilage. The proliferation of PJAC within 3 months of transplantation was stronger than that of adult cartilage. The enhanced expression of Lin28 may be one of the mechanisms by which PJAC achieved stronger proliferation ability than adult cartilage. PJAC technology has shown good application prospects for repairing cartilage defects.

Donor Cell Fate in Particulated Juvenile Allograft Cartilage for the Repair of Articular Cartilage Defects.

BACKGROUND: Particulated juvenile allograft cartilage (PJAC) has demonstrated good clinical efficacy in repairing articular cartilage defects, but the related repair mechanism after transplant and the biological characteristics of the transplanted cells are still unclear. PURPOSE: To study the efficacy of PJAC in repairing full-thickness cartilage defects and the specific fate of donor cells to provide experimental evidence for its clinical application. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty female Guizhou minipigs were randomly divided into an experimental group and a control group. An 8-mm cylindrical full-thickness cartilage defect was created in the femoral trochlea of 1 knee in all minipigs. The experimental group received transplant of PJAC from 5 male juvenile Guizhou minipigs (PJAC group; n = 10) and the control group received autologous cartilage chips (ACC group; n = 10). Follow-up assessments were conducted at 1 month and 3 months to track the transplanted cells by the male-specific sex-determining region Y-linked (SRY) gene; tissue sections were hybridized in situ, and O'Driscoll histological scoring was performed according to hematoxylin and eosin staining, safranin O and fast green staining, and toluidine blue O staining, as well as immunohistochemical evaluation of aggrecan and Sry-type HMG-box 9 (SOX9). RESULTS: All 20 Guizhou minipigs were followed; no infection or incision healing disorder occurred after the operation. By SRY in situ hybridization, the SRY signal of the transplanted cells was positive in the repaired tissue of the defect, and the SRY positive signal could still be detected in repaired tissue at 3 months postoperatively. The average number of positive cells was 68.6 ± 11.91 at 1 month and 32.6 ± 3.03 at 3 months (confocal microscope: ×400), and the difference was statistically significant. The O'Driscoll histological scores were 14 ± 0.71 in the ACC group and 9.8 ± 0.84 in the PJAC group at 1 month, and 18 ± 1.20 in the ACC group and 17.4 ± 1.14 in the PJAC group at 3 months. The scores were statistically significant between the ACC group and PJAC group at 1 month. The positive rates of SOX9 in the PJAC and ACC groups at 1 month were 67.6% ± 3.78% and 63.4% ± 5.30%, respectively, and the difference was not statistically significant (P > .05). The positive rates of SOX9 in the PJAC and ACC groups at 3 months were 68.8% ± 2.69% and 17.1% ± 1.26%, respectively, and the difference was statistically significant (P < .05). The positive rates of aggrecan in the PJAC and ACC groups at 1 month were 40.5% ± 2.78% and 42.4% ± 0.54% respectively, and the difference was not statistically significant (P > .05). The positive rates of aggrecan in the PJAC and ACC groups at 3 months were 40.8% ± 1.50% and 30.1% ± 2.44%, respectively, and the difference was not statistically significant (P > .05). CONCLUSION: An animal model was established with Guizhou minipigs, and the cartilage defect was repaired with PJAC from male minipigs. The SRY gene positive signal could be detected from the repaired tissue by in situ hybridization, indicating that the transplanted cells survived at least 3 months. The key genes of cartilage formation, SOX9 and aggrecan, were expressed at 1 month and 3 months, and SOX9 expression was stronger in the PJAC group than the ACC group at 3 months. CLINICAL RELEVANCE: This study suggests that it is feasible to study the biological characteristics of transplanted cells in the cartilage region by the sex-determining gene.

[Comparison of early effectiveness and safety of simultaneous and staged bilateral unicompartmental knee arthroplasty for bilateral anteromedial compartment osteoarthritis].

OBJECTIVE: To compare the early effectiveness and safety of simultaneous bilateral and staged bilateral unicompartmental knee arthroplasty (UKA) in treatment of anteromedial compartment osteoarthritis. METHODS: The clinical data of 31 patients with bilateral anteromedial compartment osteoarthritis who underwent bilateral UKAs between January 2015 and January 2017 was retrospectively analyzed. Of them, 17 patients were treated with simultaneous bilateral UKAs (simultaneous group) and 14 patients with staged bilateral UKAs (staged group). There was no significant difference in gender, age, body mass index, osteoarthritis grading, and preoperative hip-knee-ankle angle, knee society score (KSS), visual analogue scale (VAS) score, and range of motion (ROM) of knee between the two groups ( P>0.05). The operation time, blood loss, hospitalization stay, minimum hemoglobin value during 10 days after operation, and hospitalization cost were recorded. The staged group was compared by the sum of two operations. The effectiveness was evaluated by KSS score, VAS score, ROM at 3, 6, 12 months after operation, and patient satisfaction scores were recorded at 12 months after operation. RESULTS: The operation time, hospitalization stay, and hospitalization cost of the simultaneous group were significantly lower than those of the staged group ( P<0.05). There was no significant difference in blood loss and the minimum hemoglobin value during 10 days after operation between the two groups ( P>0.05). Superficial infection occurred in 1 side of 1 case (7.1%) in staged group. Postoperative delirium occurred in 1 case (5.9%) in simultaneous group. There was no significant difference in incidence of postoperative complications between the two groups ( P=1.000). Patients in both groups were followed up 12-32 months (mean, 24.7 months). There was no significant difference in KSS score between the two groups at 3 months after operation ( t=0.896, P=0.392). KSS scores were significanly higher in simultaneous group than in staged group at 6 and 12 months after operation ( P<0.05). There was no significant difference in ROM and VAS scores between the two groups at 3, 6, and 12 months after operation ( P>0.05). At 12 months after operation, the patient satisfaction scores were significantly higher in simultaneous group than in staged group ( P<0.05). X-ray films showed no loosening of the prosthesis in the two groups. CONCLUSION: Simultaneous bilateral UKAs has the same security as staged bilateral UKAs. Meanwhile knee function recovery was better, hospitalization stay and hospitalization cost reduced, and patient satisfaction was higher in simultaneous bilateral UKAs.

The Use of Particulated Juvenile Allograft Cartilage for the Repair of Porcine Articular Cartilage Defects.

BACKGROUND: The repair of porcine articular cartilage defects by using particulated juvenile allograft cartilage (PJAC) has demonstrated good short-term clinical efficacy, but the repair process and mechanism have not been fully elucidated. PURPOSE: To study the efficacy of PJAC in repairing full-thickness cartilage defects and to provide an experimental basis for its clinical application. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty Guizhou minipigs were randomly divided into an experimental group and control group. An 8-mm cylindrical full-thickness cartilage defect was created in the femoral trochlea of either knee in all minipigs. The experimental group received the PJAC transplantation (PJAC group; n = 15) and the control group received autologous cartilage chips (ACC group; n = 15). Five minipigs were euthanized at 1, 3, and 6 months in each group to obtain samples, which were evaluated by general view of the knee joint and histomorphometry of the chondral defect area (hematoxylin and eosin, safranin O). International Cartilage Repair Society (ICRS) II semiquantitative evaluation and collagen type II staining immunohistochemistry were also performed. RESULTS: All 30 Guizhou minipigs were followed; there was no infection or incision healing disorder after the operation. At 1 month postoperatively, more hyaline cartilage was found in the ACC group (29.4%) compared with the PJAC group (20.1%) (P < .05); there was no statistical difference between the 2 groups at 3 and 6 months after operation. The fibrocartilage content in the ACC group was significantly more than that in the PJAC group at 1 and 3 months postoperatively (27.4% vs 18.2% and 49.9% vs 41.1%, respectively; P < .05); significant differences disappeared at 6 months postoperatively. The PJAC group produced more fibrous tissue than the ACC group at 1 and 3 months postoperatively (60.1% vs 40.6% and 38.8% vs 24.4%, respectively; P < .05) but showed no statistical difference at 6 months postoperatively. Regarding the ICRS II scores, those of the ACC group were significantly better than the scores of the PJAC group in some subclasses at 3 and 6 months postoperatively. The positive rates of immunohistochemical staining in the ACC group were higher at 1 and 3 months postoperatively than those in the PJAC group (54.2% vs 37.8% and 46.4% vs 34.4%, respectively; P < .05). The difference was not statistically significant between the 2 groups at 6 months postoperatively. CONCLUSION: Both PJAC and ACC can produce a good repair effect on cartilage defects. At 1 and 3 months postoperatively, ACC resulted in better outcomes than PJAC, but there was no statistical difference in the repair effect between the 2 techniques at 6 months postoperatively. CLINICAL RELEVANCE: Based on this animal experiment, further clinical studies are needed to investigate PJAC as a possible alternative first-line treatment for cartilage defects.