Protocol for extracting and isolating porcine bone-marrow-derived macrophages from ribs.

Due to anatomical and biological similarities with humans, pigs are increasingly used for inflammation- and immune-related studies in biomedical research, including the field of osteonecrosis and osteoimmunology. Here, we present a protocol for rib extraction, isolation of the bone marrow by centrifugation, and processing to obtain bone-marrow-derived macrophages (BMDMs). Then, we describe the procedures of in vitro experiments to evaluate the cell phenotype. For complete details on the use and execution of this protocol, please refer to Andre et al.1.

Rib Cage Morphogenesis in the Human Embryo: A Detailed Three-Dimensional Analysis.

Formation of the skeletal structure in the human embryo has important consequences in terms of support, protection, and function of organs and other systems. We aimed to describe the formation of the rib cage during the embryonic period, in order to detect prominent features and identify the possible factors affecting rib cage morphology. We employed high-resolution digitized imaging data (n = 34) obtained in human embryos with Carnegie stage (CS) between 17 and 23. The rib cage became detectable as cartilage formation at CS17, expanding outward from the dorsal side of the chest-abdominal region. Ribs elongated progressively to surround the chest, differentiating into the upper and lower rib cage regions by CS20. The ends of corresponding ribs in the upper region elongated toward each other, leading to their joining and sternum formation between CS21 and CS23, while the lower region of the rib cage remained widely open. The rib cage area with the largest width shifted from the 5th rib pair at CS17 to the 9th pair at CS23. The depth of the rib cage was similar across the upper region at CS17, with the major portion remaining in the middle part after CS20. The heart was located beneath the rib pairs providing the largest depth, while the liver was located beneath the rib pairs providing the largest width. Formation of the sternum, development of spinal kyphosis, and organization of larger internal organs within the thoracic and abdominal cavity are possible factors affecting rib cage morphology. Anat Rec, 302:2211-2223, 2019. © 2019 American Association for Anatomy.

Utility of a rotation/revolution-type agitator for chondrocyte isolation during preparation of engineered cartilage.

During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1 mm3-thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1 mg/mL liberase TH (collagenase/thermolysin) at 37 °C for 4 h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering.

Size and Proportions of Slow-Twitch and Fast-Twitch Muscle Fibers in Human Costal Diaphragm.

Smaller diaphragmatic motor unit potentials (MUPs) compared to MUPs of limb muscles lead to the hypothesis that diaphragmatic muscle fibers, being the generators of MUPs, might be also smaller. We compared autopsy samples of costal diaphragm and vastus lateralis of healthy men with respect to fibers' size and expression of slow myosin heavy chain isoform (MyHC-1) and fast 2A isoform (MyHC-2A). Diaphragmatic fibers were smaller than fibers in vastus lateralis with regard to the mean minimal fiber diameter of slow-twitch (46.8 versus 72.2 µm, p < 0.001), fast-twitch (45.1 versus 62.4 µm, p < 0.001), and hybrid fibers (47.3 versus 65.0 µm, p < 0.01) as well as to the mean fiber cross-sectional areas of slow-twitch (2376.0 versus 5455.9 µm2, p < 0.001), fast-twitch (2258.7 versus 4189.7 µm2, p < 0.001), and hybrid fibers (2404.4 versus 4776.3 µm2, p < 0.01). The numerical proportion of slow-twitch fibers was higher (50.2 versus 36.3%, p < 0.01) in costal diaphragm and the numerical proportion of fast-twitch fibers (47.2 versus 58.7%, p < 0.01) was lower. The numerical proportion of hybrid fibers did not differ. Muscle fibers of costal diaphragm have specific characteristics which support increased resistance of diaphragm to fatigue.

The Role of Cell Seeding, Bioscaffolds, and the In Vivo Microenvironment in the Guided Generation of Osteochondral Composite Tissue.

Tissue engineering based on cell seeding, bioscaffolds, and growth factors has been widely applied for the reconstruction of tissue defects. Recent progress has fueled in vivo tissue engineering techniques in becoming hot topics in regenerative medicine and reconstructive surgery. To improve the efficacy of tissue engineering, we here investigated the roles of cell seeding, bioscaffolds, growth factors, and in vivo microenvironment (IM) in tissue regeneration. Bone marrow-derived stem cells, allogeneic demineralized bone matrix as bioscaffold, and growth factor bone morphogenetic protein 2/transforming growth factor, and the IM of rib periosteum and perichondrium were used in different combinations for the generation of osteochondral composite tissue. Self-regenerated neocomposite tissue based on the IM alone exhibited excellent anatomical configuration, vascularization, biomechanical stability, and function similar to native controls. Our findings indicate that the IM is a crucial factor in biofunctional tissue generation. Further refinement and development of this technique may enable transfer to clinical application with broad spectrum of application.

Laser radiation effect on chondrocytes and intercellular matrix of costal and articular cartilage impregnated with magnetite nanoparticles.

BACKGROUND AND OBJECTIVE: Magnetic nanoparticles with the ability to absorb laser radiation are the perspective agents for the early diagnostics and laser therapy of degenerative cartilage. The effect of starch stabilized magnetite nanoparticles (SSNPs) on the cartilage structure components has never been studied before. The aim of the work is to establish the Erbium:glass laser effect on costal and articular cartilage impregnated with SSNPs. MATERIALS AND METHODS: Porcine articular and costal cartilage disks (2.0 mm in diameter and 1.5-2 mm in thickness) were impregnated with SSNPs and irradiated using a 1.56 µm laser in therapeutic laser setting. The one sample group underwent the second irradiation after the SSNPs impregnation. The samples were analyzed by the means of histology, histochemistry and transmission electron microscopy (TEM) to reveal the alterations of cells, glycosaminoglycans and collagen network. RESULTS: The irradiated cartilage demonstrates the higher content of cell alterations than the intact one due to the heat and mechanical affection in the course of laser irradiation. However the alterations are localized at the areas near the irradiated surfaces and not dramatic. The impregnation of SSNPs does not cause any additional cell alterations. For both costal and articular cartilage the matrix alterations of irradiated samples are not critical: there is the slight decrease in acid proteoglycan content at the irradiated areas while the collagen network is not altered. Distribution and localization of impregnated SSNPs is described: agglomerates of 150-230 nm are observed located at the borders between matrix and cell lacunas of articular cartilage; SSNPs of 15-45 nm are found in the collagen network of costal cartilage. CONCLUSIONS: It was shown that SSNPs do not appreciably affect the structural components of both articular and costal cartilage and can be safely used for the laser diagnostics and therapy. The area of structural alterations is diffuse and local as the result of the mechanical and heat effect of laser impact. SSNPs reveal the areas of the structural alterations of cartilage matrix and give information about the size of the pores and defects.

Hyperspectral multimodal CARS microscopy in the fingerprint region.

A simple scheme for multimodal coherent anti-Stokes Raman scattering (CARS) microscopy is based on the spectral focusing of ultrafast-oscillator-derived pump/probe light and synchronous photonic crystal fiber (PCF) fiber-generated broadband Stokes light. To date, such schemes allowed rapid hyperspectral imaging throughout the CH/OH high frequency region (2700-4000 cm(-1) ). Here we extend this approach to the middle (1640-3300 cm(-1) ) and fingerprint regions (850-1800 cm(-1) ) of the Raman spectrum. Our simple integrated approach to rapid hyperspectral CARS microscopy in the fingerprint region is demonstrated by applications to label-free multimodal imaging of cellulose and bulk bone, including use of the phosphate resonance at 960 cm(-1) .

Inducing articular cartilage phenotype in costochondral cells.

INTRODUCTION: Costochondral cells may be isolated with minimal donor site morbidity and are unaffected by pathologies of the diarthrodial joints. Identification of optimal exogenous stimuli will allow abundant and robust hyaline articular cartilage to be formed from this cell source. METHODS: In a three factor, two level full factorial design, the effects of hydrostatic pressure (HP), transforming growth factor ß1 (TGF-ß1), and chondroitinase ABC (C-ABC), and all resulting combinations, were assessed in third passage expanded, redifferentiated costochondral cells. After 4 wks, the new cartilage was assessed for matrix content, superficial zone protein (SZP), and mechanical properties. RESULTS: Hyaline articular cartilage was generated, demonstrating the presence of type II collagen and SZP, and the absence of type I collagen. TGF-ß1 upregulated collagen synthesis by 175% and glycosaminoglycan synthesis by 75%, resulting in a nearly 200% increase in tensile and compressive moduli. C-ABC significantly increased collagen content, and fibril density and diameter, leading to a 125% increase in tensile modulus. Hydrostatic pressure increased fibril diameter by 30% and tensile modulus by 45%. Combining TGF-ß1 with C-ABC synergistically increased collagen content by 300% and tensile strength by 320%, over control. No significant differences were observed between C-ABC/TGF-ß1 dual treatment and HP/C-ABC/TGF-ß1. CONCLUSIONS: Employing biochemical, biophysical, and mechanical stimuli generated robust hyaline articular cartilage with a tensile modulus of 2 MPa and a compressive instantaneous modulus of 650 kPa. Using expanded, redifferentiated costochondral cells in the self-assembling process allows for recapitulation of robust mechanical properties, and induced SZP expression, key characteristics of functional articular cartilage.

Ift88 regulates Hedgehog signaling, Sfrp5 expression, and ß-catenin activity in post-natal growth plate.

Primary cilia are present on most cell types including chondrocytes. Dysfunction of primary cilia results in pleiotropic symptoms including skeletal dysplasia. Previously, we showed that deletion of Ift88 and subsequent depletion of primary cilia from chondrocytes resulted in disorganized columnar structure and early loss of growth plate. To understand underlying mechanisms whereby Ift88 regulates growth plate function, we compared gene expression profiles in normal and Ift88 deleted growth plates. Pathway analysis indicated that Hedgehog (Hh) signaling was the most affected pathway in mutant growth plate. Expression of the Wnt antagonist, Sfrp5, was also down-regulated. In addition, Sfrp5 was up-regulated by Shh in rib chondrocytes and regulation of Sfrp5 by Shh was attenuated in mutant cells. This result suggests Sfrp5 is a downstream target of Hh and that Ift88 regulates its expression. Sfrp5 is an extracellular antagonist of Wnt signaling. We observed an increase in Wnt/ß-catenin signaling specifically in flat columnar cells of the growth plate in Ift88 mutant mice as measured by increased expression of Axin2 and Lef1 as well as increased nuclear localization of ß-catenin. We propose that Ift88 and primary cilia regulate expression of Sfrp5 and Wnt signaling pathways in growth plate via regulation of Ihh signaling.

The phenotypic characterization of A13/BACii, a novel bovine chondrocytic cell line with differentiation potential.

In cartilage research bovine articular cartilage is used as an alternative to human tissue. However, animal material is subject to availability and primary cultures undergo senescence, limiting their use. Here we report the immortalization of primary bovine chondrocytes, which could be used as a surrogate for freshly isolated chondrocytes. Chondrocytes were isolated from cartilage explants and immortalized using 1.0 µg/ml benzo[alpha]pyrene. For 3-dimensional culture, chondrocytes were resuspended in 0.5% low-melt agarose at high density (HD) and cultured for 24 h prior to determining changes in expression profile and morphology. A13/BACii chondrocytes acquired a 'flat' irregular morphology and a foetal-like cell volume (1,509.59 ± 182.04 µm(3)). The human cell line C-20/A4 showed a statistically similar volume and length to A13/BACii. Two-dimensional-cultured A13/BACii expressed elevated levels of type I collagen (col1), reduced levels of type II collagen (col2) compared to freshly isolated chondrocytes and an overall col2 to col1 expression ratio (col2:col1) of 0.11 ± 0.01. Upon 3-dimensional encapsulation, there was a significant rise in col2 expression in both A13/BACii and C-20/A4, suggesting a capacity for redifferentiation in both cell lines with a return of col2:col1 values of A13/BACii to values previously observed in primary chondrocytes. A13/BACii chondrocytes expressed aggrecan, matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, further supporting indications of the differentiated phenotype. Here we report the creation of a novel chondrocytic cell line and demonstrate its strong potential for redifferentiation upon HD 3-dimensional encapsulation, providing an alternative to conventional dedifferentiated cell lines and primary culture.

Influence of abutment design on the success of immediately loaded dental implants: experimental and numerical studies.

The aim of the present study was to investigate experimentally and numerically the influence of a fine threaded- against a roughened-cervical region of immediately loaded dental implants in combination with straight and 20°-angled abutments on the implant primary stability. A total of 30 implants were inserted in bovine rib-segments, 14 cervically roughened implants and 16 implants with fine cervical threads. Each implant system received two abutments, straight and 20°-angled. Implant displacements and rotations were measured using a biomechanical measurement system. Subsequently, eight samples were selected for geometrical reconstruction and numerical investigation of stress and strain distributions in the bone by means of the finite element method. Experimentally, both implant systems showed similar behaviour with the straight abutments concerning displacements and rotations. However, fine threaded implants showed much less displacement and rotation against roughened implants when angled abutments were considered. Numerically, stresses were within 35-45 MPa in the cortical bone for both implant systems. The strains showed highest values within the spongious bone with the roughened implants connected to angled abutments. The results indicate that implants with fine cervical threads could be recommended in particular with angled abutments. The outcomes of this study are currently confirmed by long-term clinical investigations.

Effects of different levels of crushing on the viability of rabbit costal and nasal septal cartilages.

BACKGROUND: Cartilage grafts are frequently used in nasal surgery for structural and/or aesthetic purposes. The literature holds contradictory reports concerning the effect of crushing on the viability of cartilage grafts. METHODS: Nasal septal and costal cartilage grafts were harvested from 12 New Zealand rabbits. Each nasal septal and costal cartilage was divided into five equal pieces. One of the pieces was left intact and the remaining four were prepared as slightly, moderately, severely, or significantly crushed. The cartilage pieces were then autoimplanted into the paravertebral skin of the rabbits. The animals were euthanized 4 months later and the effect of crushing on cartilage grafts was assessed pathologically. RESULTS: The viability of the chondrocytes was found to be decreased as the level of crushing increased. The mean chondrocyte viability rates for the intact, slightly crushed, moderately crushed, severely crushed, and significantly crushed cartilages were 88, 75, 51, 41, and 13 percent for the septal cartilages and 94, 83, 62, 32, and 26 percent for the costal cartilages, respectively. The differences between the mean viability rates of septal and costal cartilage groups were statistically not significant. CONCLUSIONS: The level of crushing determines the rate of viability for the crushed cartilage. Viability rates and the clinical properties of the slightly crushed cartilage grafts at long-term follow-up may be similar to those of the intact cartilage grafts. However, severe or significant crushing leads to a decrement in the viability of the chondrocytes and may cause unpredictable degrees of volume loss at long-term follow-up.

Analysis of the effect of osteon diameter on the potential relationship of osteocyte lacuna density and osteon wall thickness.

An important hypothesis is that the degree of infilling of secondary osteons (Haversian systems) is controlled by the inhibitory effect of osteocytes on osteoblasts, which might be mediated by sclerostin (a glycoprotein produced by osteocytes). Consequently, this inhibition could be proportional to cell number: relatively greater repression is exerted by progressively greater osteocyte density (increased osteocytes correlate with thinner osteon walls). This hypothesis has been examined, but only weakly supported, in sheep ulnae. We looked for this inverse relationship between osteon wall thickness (On.W.Th) and osteocyte lacuna density (Ot.Lc.N/B.Ar) in small and large osteons in human ribs, calcanei of sheep, deer, elk, and horses, and radii and third metacarpals of horses. Analyses involved: (1) all osteons, (2) smaller osteons, either ≤150 µm diameter or less than or equal to the mean diameter, and (3) larger osteons (>mean diameter). Significant, but weak, correlations between Ot.Lc.N/B.Ar and On.W.Th/On.Dm (On.Dm = osteon diameter) were found when considering all osteons in limb bones (r values -0.16 to -0.40, P < 0.01; resembling previous results in sheep ulnae: r = -0.39, P < 0.0001). In larger osteons, these relationships were either not significant (five/seven bone types) or very weak (two/seven bone types). In ribs, a negative relationship was only found in smaller osteons (r = -0.228, P < 0.01); this inverse relationship in smaller osteons did not occur in elk calcanei. These results do not provide clear or consistent support for the hypothesized inverse relationship. However, correlation analyses may fail to detect osteocyte-based repression of infilling if the signal is spatially nonuniform (e.g., increased near the central canal).

Podoplanin is expressed by a sub-population of human foetal rib and knee joint rudiment chondrocytes.

The aim of this study was to determine if podoplanin was expressed by rudiment chondrocytes in human foetal cartilages. Podoplanin was immunolocalised in first trimester human foetal rib and knee joint rudiments to a sub-population of chondrocytes deep in the rib rudiments, tibial and femoral growth plates and cells associated with the cartilage canals of the foetal knee joint rudiments. Lymphatic vessels in the loose stromal tissues surrounding the developing rudiments were also demonstrated on the same histology slides using antipodoplanin (MAb D2-40) and anti-LYVE-1 and differentiated from CD-31 positive blood vessels confirming the discriminative capability of the antibody preparations used. The D2-40 positive rib and knee rudiment chondrocytes were not stained with antibodies to LYVE-1, CD-31 or CD-34 however perlecan was a prominent pericellular proteoglycan around these cells confirming their chondrogenic phenotype. Discernable differences were evident between the surface and deep rudiment chondrocytes in terms of their antigen reactivities detected with MAb D2-40 or antiperlecan antibodies. Binding of the cytoplasmic tail of PDPN to the ERM proteins ezrin, radixin and moeisin may result in changes in cytoskeletal organisation which alter the phenotype of this central population of rudiment cells. This may contribute to morphological changes in the rudiment cartilages which lead to establishment of the primary ossification centres and is consistent with their roles as transient developmental scaffolds during tissue development.

An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia.

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype.

A simple in vitro culture system for tracheal cartilage development.

Semi-circular tracheal cartilage is a critical determinant of maintaining architectural integrity of the respiratory airway. The current effort to understand the morphogenesis of tracheal cartilage is challenged by the lack of appropriate model systems. Here we report an in vitro tracheal cartilage system using embryonic tracheal­lung explants to recapitulate in vivo tracheal cartilage developmental processes. With modifications of a current lung culture protocol, we report a consistent in vitro technique of culturing tracheal cartilage from primitive mouse embryonic foregut for the first time. This tracheal culture system not only induces the formation of tracheal cartilage from the mouse embryonic foregut but also allows for the proper patterning of the developed tracheal cartilage. Furthermore, we show that this culture technique can be applied to culturing other types of cartilage in vertebrae, limbs, and ribs. We believe that this novel application of our in vitro culture system will facilitate the manipulation of cartilage development under various conditions and thus enabling us to advance our current limited knowledge on cartilage biology and development.

Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis.

Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and is highly expressed in chondrocytes. In this study, we generated Bbf2h7(-/-) mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7(-/-) chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7-Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking.

Spatial gradients of blood vessels and hematopoietic stem and progenitor cells within the marrow cavities of the human skeleton.

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34(+) and CD117(+) hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34(+) or CD117(+) HSPC to the nearest trabecular surface was measured and binned in 50-microm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34(+) HSPC population, 60% of the CD117(+) HSPC population, and 72% of the BVFs were found within 100 microm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche.

Paracrine effect of transplanted rib chondrocyte spheroids supports formation of secondary cartilage repair tissue.

The study's objective was to investigate if transplanted chondrocyte or periosteal cell spheroids have influence on ingrowing bone marrow-derived cells in a novel cartilage repair approach in miniature pigs. Autologous rib chondrocytes or periosteal cells were cultured as spheroids and press-fitted into cavities that were milled into large, superficial chondral lesions of the patellar joint surface. Within the milled cavities, the subchondral bone plate was either penetrated or left intact (full-thickness or partial-thickness cavities). The transplantation of chondrocyte spheroids into full-thickness cavities induced the formation of additional secondary repair cartilage that exceeded the original volume of the transplanted spheroids. The resulting continuous tissue was rich in proteoglycans and stained positive for type II collagen. Cell labeling revealed that secondarily invading repair cells did not originate from transplanted spheroids, but rather from arroded bone marrow. However, secondary invasion of repair cells was less pronounced following transplantation of periosteal cells and absent in partial-thickness cavities. According to in vitro analyses, these observations could be ascribed to the ability of chondrocyte spheroids to secrete relevant amounts of bone morphogenetic protein-2, which was not detected for periosteal cells. Transplanted chondrocyte spheroids exert a dual function: they provide cells for the repair tissue and have a stimulatory paracrine activity, which promotes ingrowth and chondrogenesis of bone marrow-derived cells.