Biomimetic Biomaterials Research Articles

Directing immunomodulation using biomaterials for endogenous regeneration.

Stem cell therapy and tissue engineering hold considerable potential for innovative and transformative strategies to repair damaged tissue form and function. Although many approaches are adopting ex vivo expanded cells for transplantation, an alternative is to manipulate the biomaterial-host interactions that recruit the patients' own stem cells endogenously for regeneration. There are several considerations in targeting the biomaterial-host interactions therapeutically, not the least of which is the biomimetic design of extracellular matrix (ECM)-mimicking materials and the administration of navigation cues and small molecules that target specific aspects of the native healing cascades to stimulate homing of endogenous stem cells and, thereafter, their expansion and differentiation. A sequence of coordinated interactions between the local niche cells and implanted biomaterials offers signals and sign posts that may instruct the cells traveling toward the injured tissues. Furthermore, stem cell function is critically influenced by extrinsic signals provided by the niche as well as by the implanted biomaterials. Novel strategies harnessing growth factors and immunological cues to design materials not only can modulate the behavior of stem cells but also can alter innate and adaptive immunity in a controlled manner. We envisage that successful and safe endogenous regeneration will involve at least three aspects, i.e., homing of sufficient stem cells, controlling cell fate determination, and blunting host immune responses to outside biomaterial devices. Improving our understanding of the biological and physicochemical signals of biomimetic biomaterials that govern immunomodulation for in situ tissue regeneration, particularly context-dependent macrophage (Mφ) polarization, will lead to a concurrent improvement in clinical outcomes.

Mesenchymal Stem Cells and their Potential for Microengineering the Chondrocyte Niche

Mesenchymal Stem Cells and their Potential for Microengineering the Chondrocyte Niche

Biodegradation, biocompatibility, and osteoconduction evaluation of collagen-nanohydroxyapatite cryogels for bone tissue regeneration.

Designing biomimetic biomaterials inspired by the natural complex structure of bone and other hard tissues is still a challenge nowadays. The control of the biomineralization process onto biomaterials should be evaluated before clinical application. Aiming at bone regeneration applications, this work evaluated the in vitro biodegradation and interaction between human bone marrow stromal cells (HBMSC) cultured on different collagen/nanohydroxyapatite cryogels. Cell proliferation, differentiation, morphology, and metabolic activity were assessed through different protocols. All the biocomposite materials allowed physiologic apatite deposition after incubation in simulated body fluid and the cryogel with the highest nanoHA content showed to have the highest mechanical strength (DMA). The study clearly showed that the highest concentration of nanoHA granules on the cryogels were able to support cell type's survival, proliferation, and individual functionality in a monoculture system, for 21 days. In fact, the biocomposites were also able to differentiate HBMSCs into osteoblastic phenotype. The composites behavior was also assessed in vivo through subcutaneous and bone implantation in rats to evaluate its tissue-forming ability and degradation rate. The cryogels Coll/nanoHA (30 : 70) promoted tissue regeneration and adverse reactions were not observed on subcutaneous and bone implants. The results achieved suggest that scaffolds of Coll/nanoHA (30 : 70) should be considered promising implants for bone defects that present a grotto like appearance with a relatively small access but a wider hollow inside. This material could adjust to small dimensions and when entering into the defect, it could expand inside and remain in close contact with the defect walls, thus ensuring adequate osteoconductivity.

Chimeric Protein Template-Induced Shape Control of Bone Mineral Nanoparticles and Its Impact on Mesenchymal Stem Cell Fate.

Protein-mediated molecular self-assembly has become a powerful strategy to fabricate biomimetic biomaterials with controlled shapes. Here we designed a novel chimeric molecular template made of two proteins, silk fibroin (SF) and albumin (ALB), which serve as a promoter and an inhibitor for hydroxyapatite (HA) formation, respectively, to synthesize HA nanoparticles with controlled shapes. HA nanospheres were produced by the chimeric ALB-SF template, whereas HA nanorods were generated by the SF template alone. The success in controlling the shape of HA nanoparticles allowed us to further study the effect of the shape of HA nanoparticles on the fate of rat mesenchymal stem cells (MSCs). We found that the nanoparticle shape had a crucial impact on the cellular uptake and HA nanospheres were internalized in MSCs at a faster rate. Both HA nanospheres and nanorods showed no significant influence on cell proliferation and migration. However, HA nanospheres significantly promoted the osteoblastic differentiation of MSCs in comparison to HA nanorods. Our work suggests that a chimeric combination of promoter and inhibitor proteins is a promising approach to tuning the shape of nanoparticles. It also sheds new light into the role of the shape of the HA nanoparticles in directing stem cell fate.

In vitro and preliminary in vivo toxicity screening of high-surface-area TiO2–chondroitin-4-sulfate nanocomposites for bone regeneration application

The goal of this study was to prepare nontoxic, biomimetic TiO2/chondroitin-4-sulfate nanocomposites with osteointegration ability for biomedical applications. Nanocomposites with higher surface area were subjected to bioactivity study and obtained bone-like layer with stoichiometric Ca/P ratio of 1.64 and 1.66. The susceptibility of nanocomposites against Staphylococcus aureus (∼16mm) and Escherichia coli (∼12mm) is favorable in preventing the risk of bone diseases and postoperative infections. Adequate swelling and degradations properties were favorably achieved to reduce the risk of nanoparticle accumulation in cell organelles. Moreover, the toxicity in AGS cell line and biocompatibility in osteoblast-like MG-63 cell line showed no significant mitochondrial damage. In addition, the in vitro expression of osteoblast inducing genes (OCN, OPN, ALP and COL 1) and their up-regulation, and 20% of increased hatching rate in preliminary in vivo (zebrafish) analysis were favorable for the nanocomposite at the ratio of 2:0.50 than pure TiO2. Hence, it can be concluded that among the prepared nanocomposites TCs.5 is a promising biomimetic biomaterial that can be used for advanced orthopedic research and other applications.

Interface Function Design and Bone-Regenerative Engineering of Biomimetic Biomaterials by Supersonic Treatment Using Electrolyzed Water

As interface design of autotransportation materials and bioactive ceramics by supersonic treatment, human teeth-originated granules and commercial hydroxyapatite (HAp) were partially dissolved in different acid solutions, such as electrolyzed water and HNO3aqueous solution to control bio-absorption, adsorption-release of bone growth factor and anti-bacterial characteristics. Human teeth were pulverized with cooling, dissolved in strongly acidic electrolyzed water (pH 2.6-3.1) or 2.0% HNO3solution to obtain demineralized dentin matrix granules. For supersonic or stirring demineralization in the acidic electrolyzed water, dissolution efficiencies (DE) were 3-12% and dentinal tubules with 1-2μm were found, while for supersonic demineralization in the HNO3solution, DE was attained to the highest value of 86%. When HAp was dissolved with stirring in the acidic electrolyzed water, DE of porous ceramics was lower than that of spherical particles due to smaller specific surface area and good crystallinity. For supersonic dissolution of porous ceramics in the acidic electrolyzed water, DE was 30% and extension of grain boundary and micro-crack were observed. Concerning supersonic irradiation of the electolyzed water to parietal bone in rats by using ultrasonic scaler, enlargement and propagation of micro-crack were recognized on the hard tissues.

Chondrocyte Culture in Three Dimensional Alginate Sulfate Hydrogels Promotes Proliferation While Maintaining Expression of Chondrogenic Markers

The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide-pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ.

Engineered Lysozyme Amyloid Fibril Networks Support Cellular Growth and Spreading

Fibrous networks assembled from synthetic peptides are promising candidates for biomimetic cell culture platforms and implantable biomaterials. The ability of the materials to reproduce physiological cell-matrix interactions is essential. However, the synthetic complexity of such systems limits their applications, thus alternative materials are desirable. Here, we design lysozyme derived amyloid fibril networks with controllable topographies, and perform a comprehensive study of the response of cultured fibroblast and epithelial cells. At high surface coverage a favorable increase in spreading and the generation of focal adhesions was observed, due to a combination of biomimetic chemistry and morphology. Their ease of synthesis, makes the nanoscale fibrils presented here ideal materials for future clinical applications whereby large volumes of biomimetic biomaterials are required. Furthermore, the surface chemistry of the fibrils is sufficient for the promotion of focal adhesions with cultured cells, eliminating the need for complex protocols for fibril decoration with bioactive moieties.

A theranostic agent to enhance osteogenic and magnetic resonance imaging properties of calcium phosphate cements

With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies.

Improved functionalization of electrospun PLLA/gelatin scaffold by alternate soaking method for bone tissue engineering

Biomimetic biomaterials are widely being explored as scaffold for bone regeneration. In this study, we prepared poly-l-lactic acid/hydroxyapatite (PLLA/HA) and poly-l-lactic acid/gelatin/hydroxyapatite (PLLA/Gel/HA) scaffold by electrospinning of poly-l-lactic acid (PLLA) and a blend of poly-l-lactic acid/gelatin (PLLA/Gel) followed by hydroxyapatite (HA) mineralization via alternate soaking in calcium and phosphate (Ca–P) solutions. HA growth on scaffold after each soaking cycle was confirmed by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The functional groups (COO and NH2) of gelatin in the PLLA/Gel scaffold facilitated the surface nucleation of HA as compared to the PLLA scaffold. Leaching study showed HA in PLLA/Gel/HA scaffold acts as binder of gelatin and eliminates use of toxic crosslinking agents. In vitro cell attachment on these scaffolds was assessed by using human osteosarcoma cells (MG-63). Cell proliferation on scaffolds was examined by MTT assay. MTT results clearly indicated that mineralized scaffolds did not inhibit the eventual cell proliferation. Alkaline phosphatase (ALP) activity of MG-63 cells was found to be the highest on PLLA/Gel/HA at day 7 compared to all other scaffolds. Complement activation study revealed minimum terminal complement complex (TCC) concentration for PLLA/Gel and PLLA/Gel/HA (617.33 and 654.13ng/mL respectively). These results demonstrate the proficiency of PLLA/Gel/HA scaffold in better osteostimulation with lesser immune response, which attributed to synergistic role of gelatin and HA. Thus, by mimicking the natural microenvironment PLLA/Gel/HA scaffolds can become the choice of material in bone tissue engineering.

Chiral and water‐soluble poly(2‐oxazoline)s

AbstractWe describe the synthesis and characterization of the first water‐soluble and chiral poly(2,4‐disubstituted‐2‐oxazoline)s. While poly(2,4‐dimethyl‐2‐oxazoline)s are water soluble up to 100 °C, aqueous solutions of poly(2‐ethyl‐4‐methly‐2‐oxazoline) exhibit a lower critical solution temperature. This is discussed in context with its constitutional isomers poly(2‐oxazoline)s and poly(2‐oxazine)s. Circular dichroism spectroscopy revealed strong Cotton effects, which are also responsive to temperature in aqueous solution. It is therefore hypothesized that structures, comparable to polyproline helices, are formed in aqueous solution. In contrast to polyproline, poly(2,4‐disubstituted‐2‐oxazoline)s are highly water soluble and therefore represent very interesting pseudo‐polypeptides that may be useful to develop responsive biomimetic biomaterials. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013

Sustained delivery of VEGF from designer self-assembling peptides improves cardiac function after myocardial infarction

Poor vascularization and insufficient oxygen supply are detrimental to the survival of residual cardiomyocytes or transplanted stem cells after myocardial infarction. To prolong and slow the release of angiogenic factors, which stimulate both angiogenesis and vasculogenesis, we constructed a novel self-assembling peptide by attaching the heparin-binding domain sequence LRKKLGKA to the self-assembling peptide RADA16. This designer self-assembling peptide self-assembled into nanofiber scaffolds under physiological conditions, as observed by atomic force microscopy. The injection of designer self-assembling peptides can efficiently provide the sustained delivery of VEGF for at least 1month. At 4weeks after transplantation, cardiac function was improved, and scar size and collagen deposition were markedly reduced in the group receiving VEGF with the LRKKLGKA scaffolds compared with groups receiving VEGF alone, LRKKLGKA scaffolds alone or VEGF with RADA16 scaffolds. The microvessel density in the VEGF with LRKKLGKA group was higher than that in the VEGF with RADA16 group. TUNEL and cleaved caspase-3 expression assays showed that the transplantation of VEGF with LRKKLGKA enhanced cell survival in the infarcted heart. These results present the tailor-made peptide scaffolds as a new generation of sustained-release biomimetic biomaterials and suggest that the use of angiogenic factors along with designer self-assembling peptides can lead to myocardial protection, sufficient angiogenesis, and improvement in cardiac function.

Focus on Materials in Biomaterials Science

Dear Readers of Advanced Biomaterials, The Deutsche Gesellschaft für Materialkunde (DGM) has a panel of experts in biomaterials (DGM Fachausschuss Biomaterialien) to address the growing interest of researchers, industry, public and politics in this important subject. This panel of experts works in subcommittees (Querschnittsthemen-Arbeitskreise) that focus for example on permanent implants, dental materials, antimicrobial biomaterials, tissue engineering, biomimetic materials or biointerfaces. A unique feature of the panel of experts is its focus on the materials issues in biomaterials science. The DGM panel of experts in biomaterials and its subcommittees are the knots of a strong biomaterials science network which has grown in Germany over the last years. As a result, several national and international projects have been generated by the DGM panel of experts. An example for this is the EU-FP7-Large Scale-Project “Artificial Vascularised Scaffolds for 3D-Tissue Regeneration – 3D-ArtiVasc” which is coordinated by the Fraunhofer-Gesellschaft and was initiated by the subcommittees Biomimetic Biomaterials and Tissue Engineering of the DGM panel of experts in biomaterials. At the beginning of this special issue of Advanced Biomaterials we present you six papers as examples of the work of distinguished members of the DGM panel of experts in biomaterials. Five of these papers are authored/co-authored by the chairpersons of the subcommittees of the DGM panel of experts in biomaterials. For example, Kluger et al. report about new polymeric networks for tissue engineering that can be rapidly created by printing processing. The paper by Keller et al. gives new and exciting insights into the failure mechanisms of UHMW-PE employed in total endoprostheses. Both authors chair the subcommittees Tissue Engineering and Biointerfaces, respectively, and are examples how the DGM panel of experts in biomaterials contributes to the development of young and early stage researchers. I invite you to participate in the next meeting of the DGM panel of experts in biomaterials on 12. April 2012 at the Rosensäle in Jena, Germany. I do hope you enjoy reading this issue of Advanced Biomaterials. Yours faithfully, Prof. Dr. Klaus D. Jandt Editor Advanced Biomaterials

The effect of duration on the simultaneous color contrast

Damage of hyaline cartilage species such as nasoseptal or joint cartilage requires proper reconstruction, which remains challenging due to the low intrinsic repair capacity of this tissue. Implantation of autologous chondrocytes in combination with a biomimetic biomaterial represents a promising strategy to support cartilage repair.The aim of this work was to assess the viability, attachment, morphology, extracellular matrix (ECM) production of human articular and nasoseptal chondrocytes cultured in vitro in porous poly(l-lactic) (PLLA) scaffolds of two selected pore sizes (100 and 200 μm).The PLLA scaffolds with 100 and 200 μm pore sizes were prepared via ternary thermally induced phase separation (TIPS) technique and analyzed using scanning electron microscopy (SEM). Articular and nasoseptal chondrocytes were seeded on the scaffold and cultures maintained for 7 and 14 days. Live/dead staining, (immuno-)histology and gene expression analysis of type II, type I collagen, aggrecan and SOX9 were performed to assess scaffold cytocompatibility and chondrocyte phenotype.The majority of both chondrocyte types survived on both scaffolds for the whole culture period. Hematoxylin-eosin (HE), alcian blue (visualizing glycosaminoglycans) stainings, immunoreactivity and gene expression of ECM proteins and cartilage marker (type II, I collagen, aggrecan, SOX9) of the chondrocyte scaffold constructs indicated that the smaller pore dimensions promoted the differentiation of the chondrocytes compared with the larger pore size.The present work revealed that the scaffold pore size is an important factor influencing chondrocyte differentiation and indicated that the scaffolds with 100 μm pores serve as a cytocompatible basis for further future modifications.

Micro- and nanoengineering approaches to control stem cell-biomaterial interactions.

As our population ages, there is a greater need for a suitable supply of engineered tissues to address a range of debilitating ailments. Stem cell based therapies are envisioned to meet this emerging need. Despite significant progress in controlling stem cell differentiation, it is still difficult to engineer human tissue constructs for transplantation. Recent advances in micro- and nanofabrication techniques have enabled the design of more biomimetic biomaterials that may be used to direct the fate of stem cells. These biomaterials could have a significant impact on the next generation of stem cell based therapies. Here, we highlight the recent progress made by micro- and nanoengineering techniques in the biomaterials field in the context of directing stem cell differentiation. Particular attention is given to the effect of surface topography, chemistry, mechanics and micro- and nanopatterns on the differentiation of embryonic, mesenchymal and neural stem cells.

Development of synthetic extracellular bone matrix on poly(L-lactic acid) substrates via electrostatic selfassembly and biomimetic mineralization technologies

AbstractBiomimetic formation of extracellular matrix (ECM) like coatings on artificial biomaterials provides a promising route to gain bioactive materials for tissue engineering applications. Organic ECM is also a template for apatite mineralization in bone tissue. This study was aimed at designing a multilayer film containing an ECM-like molecule on biomaterial surfaces to direct apatite mineralization and to develop biomimetic biomaterials for bone tissue engineering. A multilayer film consisting of gelatin and poly(ethylenimine) molecules was formed on a poly(L-lactic acid) substrate via electrostatic self-assembly technique. Bonelike apatite coating was formed on the multilayer film modified poly(L-lactic acid) substrate in simulated body fluids (SBFs). The amount of forming apatite could be controlled by the Ca2+ and HPO4 2- concentrations in SBF and also through the mineralization time. Gelatin terminated multilayer film can induce the apatite formation with a weak orientation in the (002) plane and the apatite is similar in composition to the natural bone. The gelatin multilayer film/apatite composite coating may serve as a promising microenvironment for bone tissue formation and growth.

The modulation of dendritic cell integrin binding and activation by RGD-peptide density gradient substrates

Dendritic cells (DCs) are central regulators of the immune system that operate in both innate and adaptive branches of immunity. Activation of DC by numerous factors, such as danger signals, has been well established. However, modulation of DC functions through adhesion-based cues has only begun to be characterized. In this work, DCs were cultured on surfaces presenting a uniform gradient of the integrin-targeting RGD peptide generated using the recently established “universal gradient substrate for click biofunctionalization” methodology. Surface expression of activation markers (costimulatory molecule CD86 and stimulatory molecule MHC-II) and production of cytokines IL-10 and IL-12p40 of adherent DCs was quantified in situ. Additionally, bound α V integrin was quantified in situ using a biochemical crosslinking/extraction method. Our findings demonstrate that DCs upregulated CD86, MHC-II, IL-10, IL-12p40 and α V integrin binding as a function of RGD surface density, with production of IL-12p40 being the marker most sensitive to RGD surface density. Surface expression of activation markers demonstrated moderate correlation with α V integrin binding, while cytokine production was highly correlated with α V integrin binding. This work demonstrates the utility of the surface density gradient platform as a high-throughput method to investigate RGD density-dependent DC adhesive responses. Furthermore, this quantitative analysis of DC integrin-based activation represents a first of its type, helping to establish the field of adhesion-based modulation of DCs as a general mechanism that has previously not been defined, and informs the rational design of biomimetic biomaterials for immunomodulation.

Designing biomaterials based on biomineralization of bone

In nature, organisms control crystal nucleation and growth using organic interfaces as templates. Scientists, in the last decades, have tried to learn from nature how to design biomimetic biomaterials inspired by the hierarchical complex structure of bone and other natural mineralised tissues or to control the biomineralization process onto biomaterials substrates to promote the osteoconductive properties of implantable devices. The design of synthetic bone analogues, i.e., with a structure and properties similar to bone, would certainly constitute a major breakthrough in bone tissue engineering. Moreover, many strategies have been proposed in the literature to develop bioactive bone-like materials, for instance using bioactive glasses. Fundamental aspects of biomineralization may be also important in order to propose new methodologies to improve calcification onto the surface of biomaterials or to develop bioactive tridimensional templates that could be used in regenerative medicine. In particular, it has been shown that some chemical groups and proteins, as well as the tridimensional matrix in which calcification would occur, play a fundamental role on the nucleation and growth of hydroxyapatite. All these distinct aspects will be reviewed and discussed in this paper.

Advance and Prospect of Bionano-Matterials

Over the past few years, bionanomaterial science has emerged as a new exciting field in which theoretical and experimental studies of structure and function of bionanomaterials have become a focus, and the importance of DNA, RNA, and peptides as bionanomaterials to the fundamental development in biology and nanomaterials has begun to be recognized. In particular, biochemistry, biophysics, biomechanics, thermodynamics, and electronic properties of DNA, RNA, and peptides, as well as intelligent composite biological materials, have become a new interdisciplinary frontier in life science and material science. There is an increasing need for a more systematic study of the basic issues involved in bionanomaterials and a more active participation of researchers in the application domain of such novel materials. Great advances have been and are being made in nanobiochip materials, nanoscale biomimetic materials, nanomotors, nanocomposite materials, interface biomaterials, and nanobiosensor and nano drug delivery systems, with enormous prospect in industrial, defense, and clinical medicine applications. Here we review some of the main advances in this field over the past few years, explore the application prospects, and discuss the concepts, issues, approaches, and challenges, with the aim of stimulating a broader interest in developing bionanomaterials technology.

Study on the Biomimetic Properties of Bone Substitute Material: Nanohydroxyapatite/Polyamide 66 Composite

To compare with the human cortical bone, the biomimetic properties of nano-hydroxyapatite/polyamide 66 composite (n-HA/PA66) were preliminarily studied qualitatively and quantitatively with TEM, XRD in crystal morphology, phase composition and crystal structure. A series of structure parameters such as cell lattice parameters (a and c), mean crystallite size and micro-strain were calculated to characterize quantitatively the microstructure of n-HA/PA66 and human cortical bone at the atomic level. The results show that n-HA/PA66 is a good biomimetic biomaterial. But there still are some differences between n-HA/PA66 and human cortical bone. Compared to human cortical bone, the crystal microstructure of n-HA/PA66 is denser, the crystal lattice of n-HA is more perfect and the distortion of crystal lattice decreases.