Human bone marrow-derived mesenchymal stem cells rescue neonatal CPAP-induced airway hyperreactivity.

Continuous positive airway pressure (CPAP) is a primary non-invasive mode of respiratory support for preterm infants. However, emerging evidence suggests CPAP could be an underlying contributor to the unintended pathophysiology of wheezing and associated airway hyperreactivity (AHR) in former preterm infants. The therapeutic benefits of mesenchymal stem cells (MSCs) have been demonstrated in a variety of animal models and several clinical trials are currently underway to assess their safety profiles in the setting of prematurity and bronchopulmonary dysplasia (BPD). In the present study, using a mouse model of neonatal CPAP, we investigated whether conditioned medium harvested from cultures of human bone-marrow derived mesenchymal stem cells (hMSC) could rescue the CPAP-induced AHR, based upon previous observations of their anti-AHR properties. Newborn mice (male and female) were fitted with a custom-made mask for delivery of daily CPAP 3 h/day for the first 7 postnatal days. At postnatal day 21 (two weeks after CPAP ended), lungs were removed, precision-cut lung slices were sectioned and incubated for 48 h in vitro in conditioned medium collected from cultures of three different hMSC donors. As expected, CPAP resulted in AHR to methacholine compared to untreated control mice. hMSC conditioned medium from the cultures of all three donors completely reversed AHR. These data reveal potential therapeutic benefits of hMSC therapy, which may be capable of rescuing the long-term adverse effects of neonatal CPAP on human airway function.

Umbilical Cord-derived Mesenchymal Stem Cells for COVID-19 Patients with Acute Respiratory Distress Syndrome (ARDS).

The coronavirus SARS-CoV-2 is cause of a global pandemic of a pneumonia-like disease termed Coronavirus Disease 2019 (COVID-19). COVID-19 presents a high mortality rate, estimated at 3.4%. More than 1 out of 4 hospitalized COVID-19 patients require admission to an Intensive Care Unit (ICU) for respiratory support, and a large proportion of these ICU-COVID-19 patients, between 17% and 46%, have died. In these patients COVID-19 infection causes an inflammatory response in the lungs that can progress to inflammation with cytokine storm, Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS), thromboembolic events, disseminated intravascular coagulation, organ failure, and death. Mesenchymal Stem Cells (MSCs) are potent immunomodulatory cells that recognize sites of injury, limit effector T cell reactions, and positively modulate regulatory cell populations. MSCs also stimulate local tissue regeneration via paracrine effects inducing angiogenic, anti-fibrotic and remodeling responses. MSCs can be derived in large number from the Umbilical Cord (UC). UC-MSCs, utilized in the allogeneic setting, have demonstrated safety and efficacy in clinical trials for a number of disease conditions including inflammatory and immune-based diseases. UC-MSCs have been shown to inhibit inflammation and fibrosis in the lungs and have been utilized to treat patients with severe COVID-19 in pilot, uncontrolled clinical trials, that reported promising results. UC-MSCs processed at our facility have been authorized by the FDA for clinical trials in patients with an Alzheimer's Disease, and in patients with Type 1 Diabetes (T1D). We hypothesize that UC-MSC will also exert beneficial therapeutic effects in COVID-19 patients with cytokine storm and ARDS. We propose an early phase controlled, randomized clinical trial in COVID-19 patients with ALI/ARDS. Subjects in the treatment group will be treated with two doses of UC-MSC (l00 × 106 cells). The first dose will be infused within 24 hours following study enrollment. A second dose will be administered 72 ± 6 hours after the first infusion. Subject in the control group will receive infusion of vehicle (DPBS supplemented with 1% HSA and 70 U/kg unfractionated Heparin, delivered IV) following the same timeline. Subjects will be evaluated daily during the first 6 days, then at 14, 28, 60, and 90 days following enrollment (see Schedule of Assessment for time window details). Safety will be determined by adverse events (AEs) and serious adverse events (SAEs) during the follow-up period. Efficacy will be defined by clinical outcomes, as well as a variety of pulmonary, biochemical and immunological tests. Success of the current study will provide a framework for larger controlled, randomized clinical trials and a means of accelerating a possible solution for this urgent but unmet medical need. The proposed early phase clinical trial will be performed at the University of Miami (UM), in the facilities of the Diabetes Research Institute (DRI), UHealth Intensive Care Unit (ICU) and the Clinical Translational Research Site (CTRS) at the University of Miami Miller School of Medicine and at the Jackson Memorial Hospital (JMH).

Sequential exposure to fibroblast growth factors (FGF) 2, 9 and 18 enhances hMSC chondrogenic differentiation.

OBJECTIVE: To test the effects of sequential exposure to FGF2, 9 and 18 on human Mesenchymal Stem Cells (hMSC) differentiation during in vitro chondrogenesis. DESIGN: Control and FGF2-expanded hMSC were cultured in aggregates in the presence of rhFGF9, rhFGF18 or rhFGFR3-specific signaling FGF variants, starting at different times during the chondroinductive program. Quantitative real time polymerase chain reaction (qRT-PCR) and immunocytochemistry were performed at different stages. The aggregate cultures were switched to a hypertrophy-inducing medium along with rhFGFs and neutralizing antibodies against FGFR1 and FGFR3. Histological/immunohistochemical/biochemical analyses were performed. RESULTS: FGF2-exposed hMSC during expansion up-regulated Sox9 suggesting an early activation of the chondrogenic machinery. FGF2, FGF9 and 18 modulated the expression profile of FGFR1 and FGFR3 in hMSC during expansion and chondrogenesis. In combination with transforming growth factor-beta (TGF-ß), FGF9 and FGF18 inhibited chondrogenesis when added at the beginning of the program (≤ d7), while exhibiting an anabolic effect when added later (≥d14), an effect mediated by FGFR3. Finally, FGFR3 signaling induced by either FGF9 or FGF18 delayed the appearance of spontaneous and induced hypertrophy-related changes. CONCLUSIONS: The stage of hMSC-dependent chondrogenesis at which the growth factors are added impacts the progression of the differentiation program: increased cell proliferation and priming (FGF2); stimulated early chondrogenic differentiation (TGF-ß, FGF9/FGF18) by shifting the chondrogenic program earlier; augmented extracellular matrix (ECM) production (FGF9/FGF18); and delayed terminal hypertrophy (FGF9/FGF18). Collectively, these factors could be used to optimize pre-implantation conditions of hMSC when used to engineer cartilage grafts.

Why are MSCs therapeutic? New data: new insight.

Adult marrow-derived mesenchymal stem cells (MSCs) are able to differentiate into bone, cartilage, muscle, marrow stroma, tendon-ligament, fat and other connective tissues. The questions can be asked, what do MSCs do naturally and where is the MSC niche? New insight and clinical experience suggest that MSCs are naturally found as perivascular cells, summarily referred to as pericytes, which are released at sites of injury, where they secrete large quantities of bioactive factors that are both immunomodulatory and trophic. The trophic activity inhibits ischaemia-caused apoptosis and scarring while stimulating angiogenesis and the mitosis of tissue intrinsic progenitor cells. The immunomodulation inhibits lymphocyte surveillance of the injured tissue, thus preventing autoimmunity, and allows allogeneic MSCs to be used in a variety of clinical situations. Thus, a new, enlightened era of experimentation and clinical trials has been initiated with xenogenic and allogeneic MSCs.

Stem cells for tissue engineering of articular cartilage.

Articular cartilage injuries are one of the most common disorders in the musculo-skeletal system. Injured cartilage tissue cannot spontaneously heal and, if not treated, can lead to osteoarthritis of the affected joints. Although a variety of procedures are being employed to repair cartilage damage, methods that result in consistent durable repair tissue are not yet available. Tissue engineering is a recently developed science that merges the fields of cell biology, engineering, material science, and surgery to regenerate new functional tissue. Three critical components in tissue engineering of cartilage are as follows: first, sufficient cell numbers within the defect, such as chondrocytes or multipotent stem cells capable of differentiating into chondrocytes; second, access to growth and differentiation factors that modulate these cells to differentiate through the chondrogenic lineage; third, a cell carrier or matrix that fills the defect, delivers the appropriate cells, and supports cell proliferation and differentiation. Stem cells that exist in the embyro or in adult somatic tissues are able to renew themselves through cell division without changing their phenotype and are able to differentiate into multiple lineages including the chondrogenic lineage under certain physiological or experimental conditions. Here the application of stem cells as a cell source for cartilage tissue engineering is reviewed.

Site-matched papillary and reticular human dermal fibroblasts differ in their release of specific growth factors/cytokines and in their interaction with keratinocytes.

The interfollicular dermis of adult human skin is partitioned into histologically and physiologically distinct papillary and reticular zones. Each of these zones contains a unique population of fibroblasts that differ in respect to their proliferation kinetics, rates at which they contract type I collagen gels, and in their relative production of decorin and versican. Here, site-matched papillary and reticular dermal fibroblasts couples were compared to determine whether each population interacted with keratinocytes in an equivalent or different manner. Papillary and reticular fibroblasts grown in monolayer culture differed significantly from each other in their release of keratinocyte growth factor (KGF) and granulocyte-macrophage colony stimulating factor (GM-CSF) into culture medium. Some matched fibroblast couples also differed in their constitutive release of interleukin-6 (IL-6). Papillary fibroblasts produced a higher ratio of GM-CSF to KGF than did corresponding reticular fibroblasts. Interactions between site-matched papillary and reticular couples were also assayed in a three-dimensional culture system where fibroblasts and keratinocytes were randomly mixed, incorporated into type I collagen gels, and allowed to sort. Keratinocytes formed distinctive cellular masses in which the keratinocytes were organized such that the exterior most layer of cells exhibited characteristics of basal keratinocytes and the interior most cells exhibited characteristics of terminally differentiated keratinocytes. In the presence of papillary dermal fibroblasts, keratinocyte masses were highly symmetrical and cells expressed all levels of differentiation markers. In contrast, keratinocyte masses that formed in the presence of reticular fibroblasts tended to have irregular shapes, and terminal differentiation was suppressed. Furthermore, basement membrane formation was retarded in the presence of reticular cells. These studies indicate that site-matched papillary and reticular dermal fibroblasts qualitatively differ in their support of epidermal cells, with papillary cells interacting more effectively than corresponding reticular cells.

Phenotypic plasticity of human articular chondrocytes.

BACKGROUND: Progenitor cells in mesenchymal tissues are important in the maintenance of tissue homeostasis and regeneration capacity. Articular cartilage is a tissue with a very low capacity for repair. One explanation could be the lack of chondrogenic progenitor cells within the adult tissue. As a test of chondrogenic differentiation potential, we examined the ability of isolated chondrocytes to take on several phenotypic identities within the mesenchymal lineage by applying culture techniques and markers used in the study of the phenotypic plasticity of marrow-derived mesenchymal stem cells (MSCs). METHODS: Culture-expanded human articular chondrocytes were analyzed for chondrogenic, adipogenic, and osteogenic capacity in defined in vitro culture systems. The osteochondrogenic potential of cells loaded into porous calcium-phosphate ceramic cubes implanted into mice was also determined. RESULTS: The different assays demonstrated that culture-expanded chondrocytes have the potential to form cartilage in pellet mass cultures, to form adipose cells in dense monolayer cultures, and to form a calcium-rich matrix in an osteogenic assay. In the in vitro assays, a variability of phenotypic plasticity was demonstrated among the donors. In contrast with MSCs, chondrocytes formed cartilage only (and not bone) in the in vivo osteochondrogenic assay. CONCLUSIONS: These results suggest that, within articular cartilage, there are chondrogenic cells that exhibit a level of phenotypic plasticity that is comparable with that of MSCs. However, there was a difference in the expression of bone in the in vivo assay.

Mesenchymal stem cells and tissue engineering for orthopaedic surgery.

Bone marrow contains multipotent mesenchymal stem cells (MSCs) that can differentiate into different mesenchymal lineages whose end-stage cells fabricate bone, cartilage, tendon, fat, and other connective tissues. Our laboratory has been focusing on the purification, culture expansion, the in vivo and the in vitro characterization of MSCs and their descendants. Given the large number of MSCs that can be generated, we have explored their use in different pre-clinical models utilizing the therapeutic potentials of MSCs for musculoskeletal disorders. Using newly evolved tissue engineering principals, we have focus on the treatment of full thickness cartilage damage and, separately, bone non-union. The experimental results suggest the MSCs may provide a powerful therapeutic tool for the treatment of musculoskeletal disorders. Future efforts should be made to establish reliable and standardized full-scale clinical approaches using MSCs for orthopaedic surgery.

[Mesenchymal stem cells--a new pathway for tissue engineering in reconstructive surgery]. / Adulte mesenchymale Stammzellen--neue Möglichkeiten der Gewebezüchtung für die plastisch-rekonstruktive Chirurgie. andreas.naumann@hno.med.uni-muenchen.de

INTRODUCTION: Mesenchymal stem cells (MSC) have the capacity to differentiate into chondrocytes with the synthesis of cartilage. This report presents the use of human adult bone marrow derived mesenchymal stem cells for tissue engineering of autologous cartilage grafts. METHODS: Human bone marrow aspirates were obtained from the iliac crest and fractionated on a Percoll gradient. The isolated hMSC were plated at 20 x 10 (6) cells per 100 mm (2) culture dish. After 21 days in culture at 37 degrees C with 5 % CO 2, the adherent multiplied MSC were trypsinized, counted, and tested for viability by trypan blue assay. The hMSCs were loaded into a sterile 15 ml polypropylene tube (0.5 Mio cells/ml) and centrifuged on the bottom of the tube at 500 g for 5 minutes. The MSC were cultivated for 3 weeks in vitro in a specific chondrogenetic medium composed of Dulbecco's Modified Eagles Medium-High Glucose supplemented with 10 ng/ml transforming growth factor-beta 1, 1 % ITS-Premix medium, 80 micro M ascorbic acid, and 100 nM dexamethasone. RESULTS: Histological and immunohistochemical studies performed after 3 weeks in three dimensional culture demonstrated the expression of cartilage specific collagen type II and X as well as proteoglycans. CONCLUSION: Human adult mesenchymal stem cells derived from bone marrow aspirates have the ability to differentiate into chondrocytes under specific culture conditions by growth factors. The use of adult mesenchymal stem cells may be a promising tool for tissue engineering of autologous cartilage grafts in reconstructive surgery in the future.

Cartilage regeneration using principles of tissue engineering.

It is well known that articular cartilage in adults has a limited ability for self-repair. Numerous methods have been devised to augment its natural healing response, but these methods generally lead to filling of the defect with fibrous tissue or fibrocartilage, which lacks the mechanical characteristics of articular cartilage and fails with time. Recently, tissue engineering has emerged as a new discipline that amalgamates aspects from biology, engineering, materials science, and surgery and that has as a goal the fabrication of functional new tissues to replace damaged tissues. The emergence of tissue engineering has facilitated the generation of new concepts and the revival of old ideas all of which has allowed a fresh approach to the repair or regeneration of tissues such as cartilage. The collaborations between scientists with different backgrounds and expertise has allowed the identification of some key principles that serve as the basis for the development of therapeutic approaches that now are less empiric and more hypothesis-driven than ever before. The current authors review some of the considerations regarding the various models used to test and validate the above repair methods and to address different aspects of the cartilage repair paradigm. Also, some key principles identified from past and current research, the need for the development of new biomaterials, and considerations in scale-up of cell-biomaterial constructs are summarized.

Tissue-engineered fabrication of an osteochondral composite graft using rat bone marrow-derived mesenchymal stem cells.

This study tested the tissue engineering hypothesis that construction of an osteochondral composite graft could be accomplished using multipotent progenitor cells and phenotype-specific biomaterials. Rat bone marrow-derived mesenchymal stem cells (MSCs) were culture-expanded and separately stimulated with transforming growth factor-beta1 (TGF-beta1) for chondrogenic differentiation or with an osteogenic supplement (OS). MSCs exposed to TGF-beta1 were loaded into a sponge composed of a hyaluronan derivative (HYAF-11) for the construction of the cartilage component of the composite graft, and MSCs exposed to OS were loaded into a porous calcium phosphate ceramic component for bone formation. Cell-loaded HYAFF-11 sponge and ceramic were joined together with fibrin sealant, Tisseel, to form a composite osteochondral graft, which was then implanted into a subcutaneous pocket in syngeneic rats. Specimens were harvested at 3 and 6 weeks after implantation, examined with histology for morphologic features, and stained immunohistochemically for type I, II, and X collagen. The two-component composite graft remained as an integrated unit after in vivo implantation and histologic processing. Fibrocartilage was observed in the sponge, and bone was detected in the ceramic component. Observations with polarized light indicated continuity of collagen fibers between the ceramic and HYAFF-11 components in the 6-week specimens. Type I collagen was identified in the neo-tissue in both sponge and ceramic, and type II collagen in the fibrocartilage, especially the pericellular matrix of cells in the sponge. These data suggest that the construction of a tissue-engineered composite osteochondral graft is possible with MSCs and different biomaterials and bioactive factors that support either chondrogenic or osteogenic differentiation.

The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate along different mesenchymal lineages including those forming bone, cartilage, tendon, fat, muscle and marrow stroma that supports hematopoiesis. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries and for hematopoietic disorders by both local and systemic application. In the present study, rat marrow-derived MSCs were ex vivo culture-expanded, labeled with (111)In-oxine, and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in excised organs, including liver, lungs, kidneys, spleen and long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected primarily in the lungs and then secondarily in the liver and other organs. When sodium nitroprusside was used, more labeled MSCs cleared the lungs resulting in a larger proportion detected in the liver. Most importantly, the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator.

Mesenchymal stem cells: building blocks for molecular medicine in the 21st century.

Mesenchymal stem sells (MSCs) are present in a variety of tissues during human development, and in adults they are prevalent in bone marrow. From that readily available source, MSCs can be isolated, expanded in culture, and stimulated to differentiate into bone, cartilage, muscle, marrow stroma, tendon, fat and a variety of other connective tissues. Because large numbers of MSCs can be generated in culture, tissue-engineered constructs principally composed of these cells could be re-introduced into the in vivo setting. This approach is now being explored to regenerate tissues that the body cannot naturally repair or regenerate when challenged. Moreover, MSCs can be transduced with retroviral and other vectors and are, thus, potential candidates to deliver somatic gene therapies for local or systemic pathologies. Untapped applications include both diagnostic and prognostic uses of MSCs and their descendents in healthcare management. Finally, by understanding the complex, multistep and multifactorial differentiation pathway from MSC to functional tissues, it might be possible to manipulate MSCs directly in vivo to cue the formation of elaborate, composite tissues in situ.

Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis.

Rat mesenchymal stem cells (rMSCs) represent a small portion of the cells in the stromal compartment of bone marrow and have the potential to differentiate into bone, cartilage, fat, and fibrous tissue. These mesenchymal progenitor cells were maintained as primary isolates and as subcultured cells in separate closed modular incubator chambers purged with either 95% air and 5% CO(2) (20% or control oxygen) or 5% oxygen, 5% CO(2), and 90% nitrogen (5% or low oxygen). At first passage, some cells from each oxygen condition were loaded into porous ceramic vehicles and implanted into syngeneic host animals in an in vivo assay for osteochondrogenesis. The remaining cells were continued in vitro in the same oxygen tension as for primary culture or were switched to the alternate condition. The first passage cells were examined for in vitro osteogenesis with assays involving the quantification of alkaline phosphatase activity and calcium and DNA content as well as by von Kossa staining to detect mineralization. Cultures maintained in low oxygen had a greater number of colonies as primary isolates and proliferated more rapidly throughout their time in vitro, as indicated by hemacytometer counts at the end of primary culture and increased DNA values for first passage cells. Moreover, rMSCs cultivated in 5% oxygen produced more bone than cells cultured in 20% oxygen when harvested and loaded into porous ceramic cubes and implanted into syngeneic host animals. Finally, markers for osteogenesis, including alkaline phosphatase activity, calcium content, and von Kossa staining, were elevated in cultures which had been in low oxygen throughout their cultivation time. Expression of these markers was usually increased above basal levels when cells were switched from control to low oxygen at first passage and decreased for cells switched from low to control oxygen. We conclude that rMSCs in culture function optimally in an atmosphere of reduced oxygen that more closely approximates documented in vivo oxygen tension.

BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis.

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF-beta 1 and BMP-2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF-beta 1 and BMP-2 on rat periosteum-derived cells during in vitro chondrogenesis in a three-dimensional aggregate culture. RT-PCR analyses for gene expression of cartilage-specific matrix proteins revealed that treatment with BMP-2 alone and combined treatment with TGF-beta 1 and BMP-2 induced time-dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP-2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF-beta 1 and BMP-2 showed no such expression. Treatment with TGF-beta 1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP-2-treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti-type II and type X collagen antibodies. In contrast, at 14 days, TGF-beta 1 + BMP-2-treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti-type X collagen antibody. Expression of BMPR-IA, TGF-beta RI, and TGF-beta RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP-2, TGF-beta 1 and TGF-beta 1 + BMP-2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP-2 induces neochondrogenesis of rat periosteum-derived cells, and that TGF-beta 1 modulates the terminal differentiation in BMP-2 induced chondrogenesis.

Monoclonal antibodies to mineralized matrix molecules of the avian eggshell.

The extracellular matrix of the mineralizing eggshell contains molecules hypothesized to be regulators of biomineralization. To study eggshell matrix molecules, a bank of monoclonal antibodies was generated that bound demineralized eggshell matrix or localized to oviduct epithelium. Immunofluorescence staining revealed several staining patterns for antibodies that recognized secretory cells: staining for a majority of columnar lining cells, staining for a minor sub-set of columnar lining cells, intensified staining within epithelial crypts, and staining of the entire tubular gland. Western blotting with the antibody Epi2 on eggshell matrix showed binding to molecules with the apparent molecular weight of eggshell matrix dermatan sulfate proteoglycan (eggshell DSPG). Immunoblots of cyanogen bromide-cleaved eggshell DSPG revealed broad band of reactivity that shifted to 25 kDa after chondroitinase digestion; indicating that the Epi2 binding site is located on a fragment which contains dermatan sulfate side chains. Immunogold labeling showed that Epi2 binds to secretory vesicles within the non-ciliated cells of the columnar epithelium, while the antibodies Tg1 and Tg2 bind to secretory vesicles of tubular gland cells. Immunogold labeling of demineralized shell matrix showed binding of Epi2, Tg1, and Tg2 to the matrix of the palisade layer, and showed little reactivity to other regions of the shell matrix. Quantification of the immunogold particles within the eggshell matrix revealed that antibodies Epi2 and Tg1 bind all calcified regions equally while antibody Tg2 has a greater affinity for the baseplate region of the calcium reserve assembly.

Hyaluronan-based polymers in the treatment of osteochondral defects.

Articular cartilage in adults has limited ability for self-repair. Some methods devised to augment the natural healing response stimulate some regeneration, but the repair is often incomplete and lacks durability. Hyaluronan-based polymers were tested for their ability to enhance the natural healing response. It is hypothesized that hyaluronan-based polymers recreate an embryonic-like milieu where host progenitor cells can regenerate the damaged articular surface and underlying bone. Osteochondral defects were made on the femoral condyles of 4-month-old rabbits and were left empty or filled with hyaluronan-based polymers. The polymers tested were ACP sponge, made of crosslinked hyaluronan, and HYAFF-11 sponge, made of benzylated hyaluronan. The rabbits were killed 4 and 12 weeks after surgery, and the condyles were processed for histology. All 12-week defects were scored with a 29-point scale, and the scores were compared with a Kruskall-Wallis analysis of variance on ranks. Untreated defects filled with bone tissue up to or beyond the tidemark, and the noncalcified surface layer varied from fibrous to hyaline-like tissue. Four weeks after surgery, defects treated with ACP exhibited bone filling to the level of the tidemark and the surface layer was composed of hyaline-like cartilage well integrated with the adjacent cartilage. At 12 weeks, the specimens had bone beyond the tidemark that was covered with a thin layer of hyaline cartilage. Four weeks after surgery, defects treated with HYAFF-11 contained a rim of chondrogenic cells at the interface of the implant and the host tissue. In general, the 12-week defects exhibited good bone fill and the surface was mainly hyaline cartilage. Treated defects received significantly higher scores than untreated defects (p < 0.05), and ACP-treated defects scored significantly higher than HYAFF-11-treated defects (p < 0.05). The introduction of these hyaluronan-based polymers into defects provides an appropriate scaffolding and favorable microenvironment for the reparative process. Further work is required to fully assess the long-term outcome of defects treated with these polymers.

Mesenchymal stem cells and gene therapy.

Multipotent human mesenchymal stem cells can be isolated from bone marrow and expanded more than 1-billion-fold in cell culture without the loss of their stem cell capacity. In addition, human mesenchymal stem cells can be transduced with genes for reporter molecules or secreted, circulating cytokines; these genes can be inserted into the genomes of mesenchymal stem cells without affecting their stem cell capacity. Thus, the stage is set for the use of mesenchymal stem cells as curative agents in genetic disorders involving skeletal tissues.